Home

Protein Thermal Shift™ Studies User Guide

1. 020 ccc eee ee eee e eee e eens 19 Set up theanalysise sals Faret detta TE ses bd he lala eet a 21 Review the well resultsu sur tt seas aatdisresst nee sendt Grut HAT ete Bld a ae ea ate 24 Review the replicate results aaararannnnnnnnnn nn rn rn eee eee e eee aes 29 M CHAPTER 2 Buffer Screening Studies eee eee eee 35 General guidelines 0c ccc aenneren enrenar enaar 36 Create and set up an experiment file for the instrument run 000200 00 eee eens 36 Prepare the protein melt reactions 0 000 e cnet eens 38 Run the protein melt reactions 000 cee ee ee eee eee ees 39 Set tp theranalySiSts lt 2c a e et ie Meet ote uk tals eter See serende 40 Review the well results o ospi esse cs ateu eek eee be ee 42 Review the replicate results 0000 e eee e eee eee 46 M CHAPTER3 Mutation Screening Studies 0 e eee eee 51 General guidelines 00 cece cece cece tte etter ee eeee 52 Create and set up an experiment file for the instrument run 00020 0 cece eee eee 52 Prepare the protein melt reactions 00 e enna 54 Run the protein melt reactions azaanannrnrnnnnn eee arane 55 Setup the analysis aura y Gens eet vee ee ete e den eer eke uante vanced eno heehee 56 Review the wellitesultS Lura rikke huker deli bide heeelt 58 Review the replicate results ununun nann cece eee seen eee 62 Protein Thermal
2. Step Ramp rate Temp C Nils 1 1 6 C s 25 0 02 00 2 0 05 C s 99 0 02 00 Melt Curve Stage O Step and Hold Continuous 99 0 C 25 0 C 1 6 C s 02 00 Step1 Step2 Dissociation Protein Thermal Shift Studies User Guide 13 Getting Started with a Protein Thermal Shift Study Create and set up an experiment file for the instrument run Create and set up an experiment file for the StepOne or StepOnePlus System 14 8 Select the optical filters for the melt curve a In the Run Method screen select the Optical Filters tab Note If you do not see the Optical Filters tab select Tools gt Preferences then select the Show optical filters for run method checkbox in the Defaults tab b In the Melt Curve Filter section select x4 580 10 m4 623 14 for the Excitation Filter Emission Filter 9 Select File gt Save then enter a file name and select a location for the experiment file Define and assign targets so you can review the melt curves for the replicate groups in the StepOne Software 1 Select Start gt All Programs gt Applied Biosystems gt StepOne Software v2 2 then log into the software Note If you are setting up the experiment file on a computer that is not connected to the instrument click Continue without Connection in the Instrument Connection Failed dialog box In the Home screen of the StepOne Software click Advanced
3. For the Passive Reference select None 5 In the Assign screen assign the target and task to wells a b For each target select the wells that contain the control protein select the Assign checkbox for Control Protein then select U for the Task Optional For no protein controls NPC select the NPC wells select the Assign checkbox for Control Protein then select N for the Task 6 Make sure that the well assignments correspond exactly with the arrangement of reactions in the reaction plate Protein Thermal Shift Studies User Guide Chapter 1 Getting Started with a Protein Thermal Shift Study Create and set up an experiment file for the instrument run For the example ligation titration study 23 24 13 14 15 16 17 18 19 20 21 22 2 3 4 5 6 7 8 3 w iT 12 A em je Je Je Je L L L L L L ee ee ee ee ee ee ee ee ee Lee Je je fo je je fr fo fo je je jer Je fo je ee ee ee eee ee ee ee Cee ee Et JE JE er Je J L 5 5 5 5 5 5 5 5 5 j5 5 J5 fe J3 jm 0 ne De De Mee Mee Mee De Mee Me Mec Mie Mee Mee Mice Mee Me Mee Mie Mee Mee DD Mee Me Mee mE E eee DEE ee eee eee eee ee DEE ee oe IMPORTANT Setup errors may result in an incorrect grouping of replicates 7 Complete the Run Method screen to define the melt curve a For the Reaction Volume Per Well enter 20 uL b For the ramp mode select Continuous c Define the thermal profile
4. None 380000 30000 20000 Huorescence E 12 00 200000 10 000 B 7 600 5000 180000 200 o oo s a a s 8 o o o amp o oS i0 100000 000 0 og i J 30 ap aa aa na son coo tm 70 28 Temperature CO a s a s a s o s 7 76 5 100 Temperature C 86 Protein Thermal Shift Studies User Guide A Supplemental Information Hardware recommendations The Protein Thermal Shift Software v1 0 is fully operational when installed on a computer with the recommended hardware configuration Component Recommended configuration Minimum requirementst Computer e 2 4 GHz CPU Intel Pentium 4 processor e 2GB of RAM or compatible processor 1 2 GHz e Disk space One hard drive no partitions 20 GB free space ee a REM Two hard drives or two partitions 300 MB free space se NE 10 GB free space on the Programs drive 20 GB free space on the data drive and 1 GB free space on the user files drive Monitor e 1280 x 1024 pixel resolution for full screen display e 1280 x 1024 pixel resolution e 16 inch color monitor for full screen display e 32 bit color e 16 inch color monitor e 32 bit color Operating Microsoft Windows 7 Operating System 32 bit Service Microsoft Windows XP system Pack 2 or later Operating System 32 bit Service or Microsoft Windows 7 Operating System 32 bit Pack 2 or later t The Minimum requirements column
5. Review the replicate results c Place the cursor over the replicate plot then wait to view a tooltip with the ATm statistics for the replicate group Note Click 2 to restore the default zoom 5 In the Replicate Groups table review the positive hits in the Hits B or Hits D column and review the ATm B or ATm D statistics in the table Protein Thermal Shift Studies User Guide 65 3 Mutation Screening Studies Review the replicate results 66 Protein Thermal Shift Studies User Guide Ligand Screening Studies Perform a ligand screening study to identify ligand candidates that stabilize a protein and lead to successful protein crystallization Perform a ligand screening study Create and set up an experiment file for the instrument run page 68 l Prepare the protein melt reactions page 70 l Run the protein melt reactions page 71 l l Review the well results page 74 l Review the replicate results page 78 Set up the analysis page 72 Example experiment files and plate template files are located in the examples folder lt drive gt Program Files Applied Biosystems Protein Thermal Shift Software examples where lt drive gt is where you installed the software To view the data for the ligand screening example used in this chapter use the Ligand_Screening_Example_ViiA7 eds and Ligand Screening Example Setup ViiA7 csv files in the ViiA7 Example Files folder Protein Thermal
6. axis for each replicate group 1 62 In the Analysis gt Replicate Results screen select lt gt Plot by then select the type of Tm statistics to review e Tm Boltzmann e Tm Derivative Specify the condition hierarchy to group the replicate plots and change the order of conditions in the plot Note Changing the condition hierarchy does not affect the results it only changes how the replicate plots are grouped and the order in which the conditions are displayed in the Replicate Results Plot a Click Condition Hierarchy above the top right corner of the plot b In the dialog box select a condition then use the Up and Down arrows to change the hierarchy of conditions The condition at the top most level of the hierarchy is displayed on the far right side of the Replicate Results Plot and the replicate plots are grouped according to the top most condition Note For the example mutation screening study set up the hierarchy so that Protein is at the bottom in the dialog box and displayed on the far left side of the Replicate Results Plot Scan the Replicate Results Plot to review the conditions that affect the Tm values relative to the reference replicate group Reference replicate plot red Sample replicate plot blue Sample replicate plot blue Protein Thermal Shift Studies User Guide Chapter 3 Mutation Screening Studies Review the replicate results 4 Review the Tm statistics for each replica
7. Inthe Analysis gt Well Results screen select Ne Show in Plot then select to show these plot components e Unselected Wells e Legend 2 Select f Color By then select Ligand to color the melt curves according to the ligand condition value assigned for the well Note To set the color for each ligand to make the plots easier to distinguish go to the Setup gt Conditions screen 3 For each replicate group select all the wells in the replicate group then review the fluorescence levels in the melt curves e For the NPC wells do you observe a rise in fluorescence If so the wells or protein melt reactions may be contaminated with protein or the dye may interact with a buffer component For the LOC wells do you observe a rise in fluorescence A rise in fluorescence in LOC wells but not in NPC wells may indicate protein contamination in the ligand or ligand dye interactions 74 Protein Thermal Shift Studies User Guide Review the well results Chapter 4 Ligand Screening Studies e For sample or reference wells do you observe flat melt curves If so condition assignments may be incorrect or a component is missing from the protein melt reactions e Within each replicate group are the fluorescence melt curves similar to each other Within each replicate group are the derivative melt curves similar to each other If the melt curves for the replicate
8. ROA start or the end line Protein Thermal Shift Studies User Guide 59 3 Mutation Screening Studies Review the well results IMPORTANT Make sure that the fluorescence at the start temperature is lower than the fluorescence at the end temperature 3 Click gt Analyze to reanalyze using the edited ROAs Note After you edit ROAs the analysis mode changes from Auto to Manual Review the flags Review the flags and Tm values then consider editing the analysis settings and or and Tm values omitting wells before you review the replicate results 1 Inthe Analysis gt Well Results screen select LE Show in Plot then select to show these plot components e Boltzmann Tm e Derivative Tm e Unselected Wells e Legend 2 Review flagged wells in the Well Table a Click the amp Flag Indicator column header to sort the wells according to the number of flags applied to the well b Scroll the table to the right to view the flags that are applied to the wells aru Fit Fr a Pe E fr fe c For flagged wells select the well in the Well Table then review the melt curves for the well compared to the other wells in the replicate group d Omit wells from analysis as necessary 3 For each replicate group select all the wells in the replicate group then review the Tm B Boltzmann Tm and the Tm D derivative Tm in the Well Table and in the melt curves Note In the melt curves the Boltzmann Tm is a green dashed
9. amp Flag Indicator column header to sort the wells according to the number of flags applied to the well b Scroll the table to the right to view the flags that are applied to the wells aru Fit Fr a Pe E fr fe c For flagged wells select the well in the Well Table then review the melt curves for the well compared to the other wells in the replicate group d Omit wells from analysis as necessary 3 For each replicate group select all the wells in the replicate group then review the Tm B Boltzmann Tm and the Tm D derivative Tm in the Well Table and in the melt curves Note In the melt curves the Boltzmann Tm is a green dashed vertical line and the derivative Tm is black dotted vertical line Are the Tm B or Tm D values significantly different from the Tm values for other wells in the replicate group Do any replicates have melt curves that are inconsistent with the other melt curves for the replicate group If so you may consider omitting wells from analysis e Are the melt curves within the replicate group similar 4 If you omitted any wells from the analysis click gt Analyze Review the 1 In the Analysis gt Well Results screen select ee Show in Plot then select to show Boltzmann fit these plot components e Boltzmann Fit e Unselected Wells e Legend 44 Protein Thermal Shift Studies User Guide 2 Review the well results Chapter 2 Buffer Screening Studies Scroll through each
10. e Boltzmann Fit e Unselected Wells e Legend Scroll through each well in the Well Table then compare the fluorescence melt curve to the Boltzmann fit curve dark green thick curve and review the value in the B Fit Boltzmann Fit column of the Well Table e How well does the fluorescence melt curve correspond to the Boltzmann fit curve e Is the B Fit value close to 1 e Is the B Fit value similar among wells in the replicate group Note When you define the ROA manually you may observe a gap between the ROA start or end temperature and the start or end of the Boltzmann fit curve The gap occurs if the defined ROA start or end temperature does not correspond exactly with a fluorescence datapoint Example well For the ViiA 7 System example ligand titration study using the Protein Thermal results Shift Control Protein and the Protein Thermal Shift Control Ligand observe the following 28 Fluorescence levels are flat in the NPC and LOC wells For the sample and reference replicate groups the fluorescence levels as displayed in the fluorescence melt curve are not significantly different so you do not need to edit the baseline The sample and reference wells contain one peak in the derivative melt curve For each replicate group the Boltzmann Tm values are similar and there are no outliers For each replicate group the derivative Tm values are similar and there are no outliers For each well the region of analysis
11. review the positive hits to identify the hits conditions that produce the maximum effect on thermal stability Replicate groups with positive hits have ATm values that exceed the threshold set in the analysis settings Note The positive hits are determined according to the analysis settings that you specified Note You must specify the reference replicate group to calculate ATm values and to determine positive hits 1 Inthe Analysis gt Replicate Results screen select lt gt Plot by then select the type of ATm statistics to review e ATm Boltzmann e ATm Derivative 2 Select the _ Show in Plot menu then select to show Positive Hits 3 Scan the green shaded area of the Replicate Results Plot for positive hits Positive hit threshold from analysis settings l Reference replicate plot ATm 0 C Positive hit Mean ATm exceeds the hit threshold Hit threshold gt 20 35 30 25 20 15 40 05 00 05 10 15 20 25 30 35 40 45 50 55 60 Temperature C _ Positive hit region Mean ATm exceeds the hit threshold 4 Review the ATm statistics for each replicate group in the Replicate Results Plot a Zoom in Click then click drag an area on the plot as many times as you need to magnify the plot 1 ath b Move the plot Click then click drag the plot until the replicate plot you want to review is in view 64 Protein Thermal Shift Studies User Guide Chapter 3 Mutation Screening Studies
12. 1 Inthe Analysis gt Well Results screen confirm that each ROA meets the following criteria For melt curves with one melt phase the curve within the ROA resembles a sigmoidal profile e At the start temperature the signal is relatively flat e Atthe end temperature the signal has already reached its maximum 2 For each replicate group edit the ROAs so that all of the wells in the replicate group have the same ROA a Select the replicates click l Define ROA in the toolbar above the melt curve plots then click drag an area in one of the plots to define a melt phase and replace the ROA Repeat for each melt phase you identify b If necessary adjust the start and end temperatures for each ROA To move the ROA Starting from within the ROA click drag the ROA e To adjust the start and end temperatures individually Click drag the ROA start or the end line IMPORTANT Make sure that the fluorescence at the start temperature is lower than the fluorescence at the end temperature 3 Click gt Analyze to reanalyze using the edited ROAs Note After you edit ROAs the analysis mode changes from Auto to Manual Review the flags Review the flags and Tm values then consider editing the analysis settings and or and Tm values omitting wells before you review the replicate results 1 Inthe Analysis gt Well Results screen select Ne Show in Plot then select to show these plot components e Boltzmann Tm e Derivative
13. 1 0 mM control ligand No protein and 0 0 mM control ligand NPC No protein and 1 0 mM control ligand LOC Required materials Required materials for protein melt reactions 18 Protein Thermal Shift Dye 1000X Protein Thermal Shift Buffer Water Protein Thermal Shift Control Protein Protein Thermal Shift Control Ligand MicroAmp Optical Reaction Plate appropriate for your real time PCR instrument MicroAmp Optical Adhesive Film appropriate for your reaction plate Protein Thermal Shift Studies User Guide Prepare the protein melt reactions Chapter 1 Getting Started with a Protein Thermal Shift Study Run the protein melt reactions We recommend that you prepare at least four replicates of each reaction 1 Prepare a fresh dilution of Protein Thermal Shift Dye 1000X to 8X 2 Place the appropriate reaction plate or tubes on ice then prepare the protein melt reactions e Make sure that the arrangement of reactions in the reaction plate corresponds exactly with the well assignments in the experiment file IMPORTANT Setup errors may result in an incorrect grouping of the data and incorrect Tm statistics e Add reaction components to the plate in the order listed Component Volume Protein Thermal Shift Buffer 5 0 uL Water 2 0 uL Protein Thermal Shift 12 5 pL Control Protein Protein Thermal Shift Control Ligand 0 mM 0 1 mM or 1mM final concentration Diluted Protein Ther
14. All Programs gt Applied Biosystems gt 7500 Software v2 0 5 to start 7500 Fast System the instrument software then log into the software 2 In the Home screen of the 7500 Software click Advanced Setup Note If you do not see the Advanced Setup button click TRA below the Design Wizard button 3 Complete the Experiment Properties screen Field Entry Experiment Name Enter a unique Experiment Name using up to 100 letters and or numbers Instrument type 7500 Fast 96 Wells or 7500 96 Wells Experiment type Melt Curve Reagent type Other Ramp speed Standard 4 In the Plate Setup gt Define Targets and Samples screen define the targets conditions In the Targets pane enter the target name for each condition select ROX for the reporter and select None for the Quencher 16 Protein Thermal Shift Studies User Guide Chapter 1 Getting Started with a Protein Thermal Shift Study 1 Create and set up an experiment file for the instrument run For the example ligand titration study Target Name T4 lig ROX None va T4lig 0 1mM L Rox None m T lig 1MM L Rox NPC Rox None Bd F Loc Rox KIKAKAKAKI M x None m M El Ji None vi 5 Define the well contents in the Plate Setup gt Assign Targets and Samples screen a In the Plate Setup screen click the Assign Targets and Samples tab b For each target cond
15. Chapter 1 Getting Started with a Protein Thermal Shift Study 1 Review the replicate results c Place the cursor over the replicate plot then wait to view a tooltip with the ATm statistics for the replicate group Note Click I to restore the default zoom 5 In the Replicate Groups table review the positive hits W in the Hits B column and review the ATm B statistics in the table Example replicate For the ViiA 7 System example ligand titration study using the starter kit observe the results following The control ligand stabilizes the protein in a concentration dependent manner The protein melt reactions that contained 1 0 mM control ligand increased the thermal stability of the control protein beyond the threshold set in the analysis settings ATm Boltzmann gt 2 0 C Next steps The use of Protein Thermal Shift studies to determine the relative thermal stability of proteins ATm values is precise and consistent with analysis using calorimetry or spectroscopy methods After you perform Protein Thermal Shift studies to screen for and identify positive hits you can validate the positive hits using calorimetry or spectroscopy methods to determine the thermodynamic stability Protein Thermal Shift Studies User Guide 33 Getting Started with a Protein Thermal Shift Study Review the replicate results 34 Protein Thermal Shift Studies User Guide Buffer Screening Studies Perform a buffer screenin
16. High NPC Low Signal and Poor Fit flags specify the condition and threshold for applying the flag Apply the analysis settings and analyze e Click Apply to apply the analysis settings and reanalyze while keeping the Analysis Settings dialog box open or e Click OK to apply the analysis settings reanalyze and close the Analysis Settings Protein Thermal Shift Studies User Guide 23 Review the well results Review the well results Getting Started with a Protein Thermal Shift Study About the melt curve plots 24 Using the Protein Thermal Shift Software review the melt curves and well table and optimize the analysis in the Analysis gt Well Results screen The Well Results screen displays fluorescence and derivative melt curve plots calculated Tm values individual well results and flags As necessary edit the analysis settings edit the baseline edit the region of analysis edit the analysis mode and omit outliers This section provides guidance on how to review and interpret the well results Some common troubleshooting causes are provided For more detailed troubleshooting information see page 83 In the Well Results screen the fluorescence data are plotted as fluorescence melt curves and as derivative melt curves The regions of analysis ROAs Boltzmann Tm values and derivative Tm values are displayed in the melt curve plots Boltzmann Tm Derivative Tm Fluorescence melt curves F Deriva
17. Make sure that the condition assignments correspond exactly with the contents of the reaction plate IMPORTANT Setup errors may result in an incorrect grouping of the data and incorrect Tm statistics For each analysis group assign the reference replicate group a Select the wells for the replicate group to use as the reference b Click RZ Assign then select Reference as the Task Click Ca Save in the toolbar to save and analyze the study Review and edit the analysis settings to optimize the analysis for your study For the examples shown in this user guide no changes were made to the analysis settings If you are reviewing the data in the example studies provided with the software try revising the analysis settings to see how the settings affect the positive hits and the flags 1 2 Click Ba Analysis Settings in the toolbar On the Positive Hit tab specify the ATm Boltzmann and ATm Derivative values to indicate a positive hit e Select gt to identify mutations that increase protein thermal stability or select lt to identify mutations that decrease protein thermal stability Enter the number of degrees C of Tm shift relative to the reference to indicate a positive hit On the Flags tab specify settings for applying flags a Select the flags to use in the analysis b For the High Background High NPC Low Signal and Poor Fit flags specify the condition and threshold for applying the flag Apply the analysis s
18. Melt Curve in the navigation pane select 7500 Software Normalized Reporter from the Plot dropdown list then select Target from the Color dropdown list 3 Review the melt curves e Do you see fluorescence signals in all of the sample wells No fluorescence signals in the sample wells may indicate missing dye or protein or an instrument problem Do you see flat fluorescence levels in the NPC wells Protein Thermal Shift Studies User Guide 55 3 Mutation Screening Studies Set up the analysis High fluorescence levels in the NPC wells may indicate protein contamination in the wells or protein melt reactions or it may indicate that the dye interacts with a component in the buffer e Do the replicates have similar melt curves 4 Save then close the experiment file Note The melt curves in the real time PCR software may not exactly match the melt curves in the Protein Thermal Shift Software When the experiment files are imported into the Protein Thermal Shift Software the Protein Thermal Shift Software reduces the noise in the fluorescence data Example melt Melt curves for the mutation screening example file from the ViiA 7 System curves Normalized Reporter Rn Temperature C mutant 1 Bf mutant 2 lec lf wid type Set up the analysis This section provides instructions for setting up the protein thermal shift study using Protein Thermal Shift Software v1 0 For more info
19. NPC select the NPC wells select the Assign checkbox for the NPC target then select N for the Task d For the passive reference select None For the example ligand titration study column 12 is empty A Hm mn 0 DN EN DNA NA NA 6 DE Hr mm mm mm EE EE ME ee DE c Hm OEE DE Eee mn DE E Hm mm 0 Im 0 NAS NAG EA 6 DE Eee jo jv c Hm Hmm mn DN NN NAS E E UD DE Oe QT Oe mm ice ju 6 Make sure that the well assignments correspond exactly with the arrangement of reactions in the reaction plate IMPORTANT Setup errors may result in an incorrect grouping of replicates 7 Complete the Run Method screen to define the melt curve a For the Reaction Volume Per Well enter 20 uL b For the ramp mode select Continuous c Define the thermal profile Time o Step Ramp rate Temp C fame 1 100 25 0 02 00 2 1 99 0 02 00 Protein Thermal Shift Studies User Guide 15 Getting Started with a Protein Thermal Shift Study Create and set up an experiment file for the instrument run Melt Curve Stage Continuous Step and Hold 99 0 C 02 00 25 0 C 100 02 00 Step 1 Step 2 8 Select File gt Save then enter a file name and select a location for the experiment file Create and set up Define and assign targets so you can review the melt curves for the replicate groups in an experiment file the 7500 Software for the 7500 or 1 Select Start gt
20. Real Time PCR System Software open the experiment file from the completed instrument run analyze and save the experiment file then review the melt curves Note You must analyze and save the experiment file in the Real Time PCR System Software before you can import it into the Protein Thermal Shift Software Some common troubleshooting causes are provided here For more detailed troubleshooting information see page 83 1 In the Home screen of the Real Time PCR System Software click Open then select the experiment file from the instrument run 2 View the melt curves Real Time PCR System Software View the melt curve ViiA 7 Software Click Analysis gt Melt Curve Plot in the navigation pane select Normalized Reporter from the Plot dropdown list then select Target from the Color dropdown list StepOne Software or 7500 Software Click Analysis gt Melt Curve in the navigation pane select Normalized Reporter from the Plot dropdown list then select Target from the Color dropdown list Melt curves for the ligand screening example file from the ViiA 7 System Normalized Reporter Rn ature C E Loc nec B Proteina 0 1mML B Proteina 0 mM L Ml Protena 1 mM L 3 Review the melt curves e Do you see fluorescence signals in all of the sample wells No fluorescence signals in the sample wells may indicate missing dye or protein or an instrument problem Do y
21. Setup Note If you do not see the Advanced Setup button click TRA below the Design Wizard button Complete the Experiment Properties screen Field Entry Experiment Name Enter a unique Experiment Name using up to 50 letters and or numbers Instrument type Select the type of instrument that you are using StepOnePlus Instrument 96 Wells or StepOne Instrument 48 Wells Experiment type Melt Curve Reagent type Other Ramp speed Fast In the Plate Setup gt Define Targets and Samples screen define the targets conditions In the Targets pane enter the target name for each condition select ROX for the reporter and select None for the Quencher For the example ligand titration study Protein 0ommL ROX vin Protein 0 1mM L ROX Yr none m Protein 1mM L ROX ov Nore m Loc ROX x none x NPC ROX none m Define the well contents in the Plate Setup gt Assign Targets and Samples screen Target Name dete dele a In the Plate Setup screen click the Assign Targets and Samples tab Protein Thermal Shift Studies User Guide Chapter 1 Getting Started with a Protein Thermal Shift Study 1 Create and set up an experiment file for the instrument run b For each target conditions select the wells with those conditions select the Assign checkbox for the target then select U for the Task c Optional For no protein controls
22. Shift Studies User Guide 67 4 Ligand Screening Studies General guidelines General guidelines For general guidelines that apply to all Protein Thermal Shift studies see page 9 Experimental Before you perform a ligand screening study we recommend that you first perform a design buffer screening study to identify a buffer in which the protein is thermally stable After you identify ligand candidates you can perform a ligand titration study to determine the optimal ligand protein ratio Replicates and For ligand screening studies we recommend that you prepare controls e At least 4 replicates of each reaction e At least 4 replicates of no protein controls NPCs e Atleast 4 replicates of ligand only controls LOCs Create and set up an experiment file for the instrument run This section contains general settings for creating and setting up an experiment file For detailed instructions see Instrument Page ViiA 7 Real Time PCR System page 12 StepOne and StepOnePlus Real Time PCR Systems page 14 7500 and 7500 Fast Real Time PCR Systems page 16 Ge file Setup Setting Experiment Experiment type Melt Curve properties Reagents Other Ramp speed Fast or Standard Target Reporter ROX properties Quencher None Plate layout Assign targets to all wells in use Passive reference None 68 Protein Thermal Shift Studies User Guide Chapter 4 Ligand Screening Studies C
23. Within each replicate group are the fluorescence melt curves similar to each other Within each replicate group are the derivative melt curves similar to each other If the melt curves for the replicates are dissimilar pipetting errors may have occurred during reaction setup or condition assignments may be incorrect 4 Ifthe derivative melt curves for the replicate group show multiple melt phases set the analysis mode to Auto Multiple Tm then review the derivative melt curves a In the Well Table or in the melt curve plot select the wells with multiple melt phases b Click Auto Analysis Options then select Auto Multiple Tm c Click gt Analyze to reanalyze using the Auto Multiple Tm analysis mode d Review the number of melt phases in the derivative melt curves e Do all replicate groups have the same number of melt phases e For each replicate group are there outliers with a different number of melt phases than the other samples in the replicate group You may consider omitting outliers from analysis Review the regions Review the ROAs detected by the software If necessary edit the ROAs of analysis ROA Note If no melt phases are detected no ROAs are defined Negative controls should have no melt phases and no ROAs 1 In the Analysis gt Well Results screen confirm that each ROA meets the following criteria e For melt curves with one melt phase the curve within the ROA resembles a sigmoidal profile e A
24. and omit outliers This section provides guidance on how to review and interpret the well results Some common troubleshooting causes are provided For more detailed troubleshooting information see page 83 Review the melt curve plots to visualize the fluorescence and derivative fluorescence data If necessary change the analysis mode Note For NPC wells the derivative melt curves are not displayed 1 Inthe Analysis gt Well Results screen select Ne Show in Plot then select to show these plot components e Unselected Wells e Legend 2 Select 8 Color By then select a buffer condition for example Buffer or Salt to color the melt curves according to the buffer condition value assigned for the well Note To set the color for each buffer condition to make the plots easier to distinguish go to the Setup gt Conditions screen Protein Thermal Shift Studies User Guide Review the well results Chapter 2 Buffer Screening Studies 3 For each replicate group select all the wells in the replicate group then review the fluorescence levels in the melt curves e For the NPC wells do you observe a rise in fluorescence If so the wells or protein melt reactions may be contaminated with protein or the dye may interact with a buffer component e For sample or reference wells do you observe flat melt curves If so condition assignments may be incorrect or a component is missing from the protein melt reactions e
25. exceeds the hit threshold 4 Review the ATm statistics for each replicate group in the Replicate Results Plot a Zoom in Click then click drag an area on the plot as many times as you need to magnify the plot 1 ath b Move the plot Click then click drag the plot until the replicate plot you want to review is in view 80 Protein Thermal Shift Studies User Guide Chapter 4 Ligand Screening Studies Review the replicate results c Place the cursor over the replicate plot then wait to view a tooltip with the ATm statistics for the replicate group Note Click 2 to restore the default zoom 5 In the Replicate Groups table review the positive hits in the Hits B or Hits D column and review the ATm B or ATm D statistics in the table Protein Thermal Shift Studies User Guide 81 4 Ligand Screening Studies Review the replicate results 82 Protein Thermal Shift Studies User Guide Troubleshooting Possible causes Symptom Recommended action Native protein has external High initial background signal page 85 The protein may not be a suitable hydrophobic sites and or a small transitional increase in candidate for Protein Thermal signal Shift studies e Perform protein dye titration studies to optimize the protein concentration and protein dye ratio Protein is heat stable and the Tm Flat signal or decrease in signal e Use an alternate method to screen exceeds the range for Pro
26. lists the lowest specifications that permit the software installation The minimum requirements may not provide optimal performance Life Technologies does not guarantee support of an installation in this environment Protein Thermal Shift Studies User Guide 87 A Supplemental Information Boltzmann fitting method Boltzmann fitting method Data from the region of analysis are fit to the Boltzmann equation to generate the Tm The Boltzmann equation F T F pre FARS fre C l e where e F T is the fluorescence at a particular temperature e F pre is the fluorescence before the transition or melting at the start of the region of analysis ROA e F post is the fluorescence after the transition or melting at the end of the ROA Tm is the melting temperature e Cis the enthalpy of the reaction 88 Protein Thermal Shift Studies User Guide Software Warranty Information Computer configuration Life Technologies supplies or recommends certain configurations of computer hardware software and peripherals for use with its instrumentation Life Technologies reserves the right to decline support for or impose extra charges for supporting nonstandard computer configurations or components that have not been supplied or recommended by Life Technologies Life Technologies also reserves the right to require that computer hardware and software be restored to the standard configuration prior to providing service or technical suppo
27. meets the recommended criteria Protein Thermal Shift Studies User Guide Chapter 1 Getting Started with a Protein Thermal Shift Study Review the replicate results Review the replicate results Using the Protein Thermal Shift Software review the Tm statistics for the replicate groups and look for positive hits in the Analysis gt Replicate Results screen This section provides guidance on how to review and interpret the replicate results Some common troubleshooting causes are provided For more detailed troubleshooting information see page 83 About the Tm ATm values are calculated for each ROA by subtracting the mean Tm for the replicate statistics group from the mean Tm for the reference replicate group If there is more than one ROA the ATm values are calculated for each ROA in the order that they appear left to right Aboutthe Replicate The Replicate Results Plot shows Tm statistics for each replicate group and Tm Results Plot datapoints for each well in the replicate group You can select to view the Boltzmann Tm statistics derivative Tm statistics ABoltzmann Tm statistics or ADerivative Tm statistics The plot shows the calculated Tm or ATm value for each sample in the replicate group The mean Tm or ATm value and the upper and lower 95 confidence limits are identified by colored diamonds red diamonds for the reference replicate groups and blue diamonds for the sample replicate groups Single Tm Analysis R
28. regulatory requirements in the following In the U S e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials e Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO CDS CSR LYO 2004 11 en Protein Thermal Shift Studies User Guide Related documentation Documentation and Support Document Catalog no Description Protein Thermal Shift Studies Ligand 4461812 Provides brief step by step procedures for performing Screening Quick Reference Protein Thermal Shift studies to screen for ligands that affect the thermal stability of the protein of interest Protein Thermal Shift Studies Buffer 4461810 Provides brief step by step procedures for performing Screening Quick Reference Protein Thermal Shift studies to screen f
29. vertical line and the derivative Tm is black dotted vertical line Are the Tm B or Tm D values significantly different from the Tm values for other wells in the replicate group Do any replicates have melt curves that are inconsistent with the other melt curves for the replicate group If so you may consider omitting wells from analysis e Are the melt curves within the replicate group similar 4 If you omitted any wells from the analysis click gt Analyze Review the 1 In the Analysis gt Well Results screen select ee Show in Plot then select to show Boltzmann fit these plot components e Boltzmann Fit e Unselected Wells e Legend 60 Protein Thermal Shift Studies User Guide Review the well results Chapter 3 Mutation Screening Studies 2 Scroll through each well in the Well Table then compare the fluorescence melt curve to the Boltzmann fit curve dark green thick curve and review the value in the B Fit Boltzmann Fit column of the Well Table e How well does the fluorescence melt curve correspond to the Boltzmann fit curve e Is the B Fit value close to 1 e Is the B Fit value similar among wells in the replicate group Note When you define the ROA manually you may observe a gap between the ROA start or end temperature and the start or end of the Boltzmann fit curve The gap occurs if the defined ROA start or end temperature does not correspond exactly with a fluorescence datapoint Example well For t
30. 0 Temperature C tego E Loc Enpe Ml protein 0 1 mL Ml Proteina 0 mL Ml proteina 1 mL Set up the analysis This section provides instructions for setting up the protein thermal shift study using Protein Thermal Shift Software v1 0 For more information about how to use the software refer to the Protein Thermal Shift Software Help Setup guidelines The experiment files that you import into the study must contain analyzed melt curve data from a complete melt curve run e Set up the analysis group so that it contains experiment files from only one instrument z Create and set up 1 In the Home screen of the Protein Thermal Shift Software click Create the study Study 2 Complete the Setup gt Properties screen e The Study Name cannot be more than 100 characters and cannot contain these characters lt gt 1 The instrument selection must match the instrument type that you used to run the protein melt reactions and generate the experiment files 72 Protein Thermal Shift Studies User Guide Review the analysis settings 8 Chapter 4 Ligand Screening Studies 4 Set up the analysis In the Setup gt Conditions screen define the conditions and condition values then list the analysis groups for your study In the Setup gt Experiment Files screen click P Import then select the experiment file eds for the instrument type that you selected for the study Repeat for e
31. 6 e Perform a buffer screening Protein Thermal Shift study to identify buffer conditions that increase thermal stability of the protein then repeat the original study using the new buffer conditions Protein Thermal Shift Studies User Guide 83 Troubleshooting Possible causes Symptom Recommended action Protein contamination in the buffer Melt curves in NPC wells Repeat the study with fresh reagents buffer component ligand or dye Mele curieedn IOC melis Protein concentration is too low Melt curves with low relative Perform protein dye titration studies fluorescence levels to optimize the protein concentration and protein dye ratio Passive reference is not set to None Flat signal or decrease in signal 1 Using your real time PCR system in the experiment file page 86 software open the experiment file 2 Set the passive reference to None e ViiA 7 Software Define screen StepOne Software or 7500 Software Plate Setup gt Assign Targets and Samples screen 3 Save and reanalyze the experiment 4 Reimport the experiment file into the Protein Thermal Shift study Incorrect filters selected with the No fluorescence or very low Repeat the protein melt reactions and ViiA 7 System fluorescence instrument run making sure that you select the correct filters when you set up the experiment file see page 12 84 Protein Thermal Shift Studies User Guide Chapter 5 Tro
32. Error standard error of the mean for the Tm value Is the value low If the value is high review the data for each replicate e Min and Max minimum and maximum Tm values for the replicate group Is the range of Tm values for the replicate group within 1 degree If the range of Tm values exceeds 1 degree review the data for each replicate 6 Omit outliers as necessary then click p Analyze Protein Thermal Shift Studies User Guide 47 2 Buffer Screening Studies Review the replicate results Review the positive In the Analysis gt Replicate Results screen review the positive hits to identify the hits conditions that produce the maximum effect on thermal stability Replicate groups with positive hits have ATm values that exceed the threshold set in the analysis settings Note The positive hits are determined according to the analysis settings that you specified Note You must specify the reference replicate group to calculate ATm values and to determine positive hits 1 Inthe Analysis gt Replicate Results screen select lt gt Plot by then select the type of ATm statistics to review e ATm Boltzmann e ATm Derivative 2 Select the _ Show in Plot menu then select to show Positive Hits 3 Scan the green shaded area of the Replicate Results Plot for positive hits Positive hit threshold from analysis settings l Reference replicate plot ATm 0 C Positive hit Mean ATm exceeds the hit threshold Hit thres
33. Plus System page 14 e 7500 or 7500 Fast System page 16 l Prepare the protein melt reactions page 18 l Run the protein melt reactions page 19 l l Review the well results page 24 l Review the replicate results page 29 Set up the analysis page 21 Protein Thermal Shift Studies User Guide 7 Getting Started with a Protein Thermal Shift Study Product information Product information Purpose of the starter kit Starter kit contents and storage The Protein Thermal Shift Starter Kit includes buffer dye a control protein and a control ligand After you follow the instructions in this chapter to learn how to perform a Protein Thermal Shift study you can continue to use the starter kit components Use the Protein Thermal Shift Buffer and the Protein Thermal Shift Dye in your protein melt reactions The buffer and dye included in the starter kit are identical to the buffer and dye in the Protein Thermal Shift Dye Kit TM The Protein Thermal Shift Starter Kit contains two boxes par Box Quantity Component Storage conditions number 4462263 Protein Sufficient for Protein Thermal Shift Buffer Room temperature RT Thermal 18 C to 25 C Shift Dye Kit 2000 reactions Protein Thermal Shift Dye Protein Thermal Shift Starter Kit Sufficient for 15 C to 25 C 100 reactions Protein Thermal Shift Control Ligand Protein Ther
34. Shift Studies User Guide 3 Contents M CHAPTER 4 Ligand Screening Studies 00cc cece eee 67 General guidelines 0 c cece cece ttt ttt teen teens 68 Create and set up an experiment file for the instrument run 2 0000000 c ee eee ees 68 Prepare the protein melt reactions 00 ccc eee nee e eens 70 Run the protein melt reactions 00 00 cee eee eee eee e teens 71 Setup the nalysis sva sasmersvdknski neser seid seder e ten ease heder kasse 72 Review the well results 0 0 cece cece ene nde een N a e ees 74 Review the replicate results unnan cece ete eee eee e eee aee 78 CHAPTER 5 Troubleshooting 0c cece cence eee eens 83 Examples of SymptOMms use sandete Gotaas ea sane ee ea ee 85 APPENDIX A Supplemental Information 0eeeeee 87 Hardware recommendations essre eero cece een n AEE ETO DRE een nee eee 87 Boltzmann fitting method 2 000 e eee e eee e ees 88 APPENDIX B Software Warranty Information 55 89 Computer configuration c 2 ccsie24 na Lacing kone Gadla keramiker ali e a i aren Dele 89 Limited product warranty 2 00000 e ence e eee e eens 89 APPENDIX C Ordering Information 0ce cece eens 91 APPENDIX D Setra he fs 93 Ghemical safety ors See eee eh rete ee 93 Biological hazard safety 0c cece eee ee ee eee eee e eee ees 94 Documentation nd Support vasse sassanid
35. Tm e Unselected Wells e Legend 2 Review flagged wells in the Well Table a Click the amp Flag Indicator column header to sort the wells according to the number of flags applied to the well b Scroll the table to the right to view the flags that are applied to the wells c For flagged wells select the well in the Well Table then review the melt curves for the well compared to the other wells in the replicate group d Omit wells from analysis as necessary Protein Thermal Shift Studies User Guide 27 1 Getting Started with a Protein Thermal Shift Study Review the well results Review the 1 Boltzmann fit For each replicate group select the wells in the replicate group then review the Tm B Boltzmann Tm and the Tm D derivative Tm in the Well Table and in the melt curves Note In the melt curves the Boltzmann Tm is a green dashed vertical line and the derivative Tm is black dotted vertical line e Are the Tm B or Tm D values significantly different from the Tm values for other wells in the replicate group Do any replicates have melt curves that are inconsistent with the other melt curves for the replicate group If so you may consider omitting wells from analysis e Are the melt curves within the replicate group similar If you omitted any wells from the analysis click gt Analyze In the Analysis gt Well Results screen select We Show in Plot then select to show these plot components
36. USER GUIDE applied biosystems by Life technologies Protein Thermal Shift Studies Using Protein Thermal Shift reagents and Protein Thermal Shift Software v1 0 Publication Part Number 4461808 Rev A Revision Date May 2011 technologies For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES LIMITED USE LABEL LICENSE RESEARCH USE ONLY The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes onl
37. ach experiment file to import into the study Note The experiment file name and data are imported Plate setup information is not imported For each experiment file assign the conditions task and analysis group to the wells that contained protein melt reactions Make sure that the condition assignments correspond exactly with the contents of the reaction plate IMPORTANT Setup errors may result in an incorrect grouping of the data and incorrect Tm statistics For each analysis group assign the reference replicate group a Select the wells for the replicate group to use as the reference b Click RZ Assign then select Reference as the Task Click Pa Save in the toolbar to save and analyze the study Review and edit the analysis settings to optimize the analysis for your study For the examples shown in this user guide no changes were made to the analysis settings If you are reviewing the data in the example studies provided with the software try revising the analysis settings to see how the settings affect the positive hits and the flags 1 2 Click Ba Analysis Settings in the toolbar On the Positive Hit tab specify the ATm Boltzmann and ATm Derivative values to indicate a positive hit e Select gt to identify ligands that increase protein thermal stability or select lt to identify ligands that decrease protein thermal stability e Enter the number of degrees C of Tm shift relative to the reference to
38. and save the experiment file in the Real Time PCR System Software before you can import it into the Protein Thermal Shift Software Some common troubleshooting causes are provided here For more detailed troubleshooting information see page 83 1 In the Home screen of the Real Time PCR System Software click Open then select the experiment file from the instrument run 2 View the melt curves Real time PCR System Software View the melt curve ViiA 7 Software Click Analysis gt Melt Curve Plot in the navigation pane select Normalized Reporter from the Plot dropdown list then select Target from the Color dropdown list StepOne Software or Click Analysis gt Melt Curve in the navigation pane select 7500 Software Normalized Reporter from the Plot dropdown list then select Target from the Color dropdown list 3 Review the melt curves e Do you see fluorescence signals in all of the sample wells No fluorescence signals in the sample wells may indicate missing dye or protein or an instrument problem Do you see flat fluorescence levels in the NPC wells Protein Thermal Shift Studies User Guide 39 2 Buffer Screening Studies Set up the analysis High fluorescence levels in the NPC wells may indicate protein contamination in the wells or protein melt reactions or it may indicate that the dye interacts with a component in the buffer e Do the replicates have similar melt curves 4 Save then
39. and the same analysis group We recommend that you include at least 4 replicates in a replicate group See region of analysis In the Protein Thermal Shift Software a study contains one or more experiment files with one or more analysis groups For each analysis group the goal of the study is to identify condition sets that have the greatest ATm affect on protein thermal stability relative to the reference In the Protein Thermal Shift Software the type of reaction performed in the well Available tasks in the software e Sample Wells with protein melt conditions that you are testing e Reference Wells with protein melt conditions to use as the basis for calculating ATm values and determining positive hits e NPC No Protein Control Wells that contain no protein See melting temperature Tm See Boltzmann Tm Tm B See derivative Tm Tm D Protein Thermal Shift Studies User Guide 99 HSSOST SEE Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit www appliedbiosystems com support technologies www lifetechnologies com
40. ate group have the same ROAs a Select the replicates click l Define ROA in the toolbar above the melt curve plots then click drag an area in one of the plots to define a melt phase and replace the ROA Repeat for each melt phase you identify b If necessary adjust the start and end temperatures for each ROA To move the ROA Starting from within the ROA click drag the ROA e To adjust the start and end temperatures individually Click drag the ROA start or the end line IMPORTANT Make sure that the fluorescence at the start temperature is lower than the fluorescence at the end temperature 3 Click gt Analyze to reanalyze using the edited ROAs Protein Thermal Shift Studies User Guide 75 4 Ligand Screening Studies Review the well results Note After you edit ROAs the analysis mode changes from Auto to Manual Review the flags Review the flags and Tm values then consider editing the analysis settings and or and Tm values omitting wells before you review the replicate results 1 Inthe Analysis gt Well Results screen select Ve Show in Plot then select to show these plot components e Boltzmann Tm e Derivative Tm e Unselected Wells e Legend 2 Review flagged wells in the Well Table a Click the amp Flag Indicator column header to sort the wells according to the number of flags applied to the well b Scroll the table to the right to view the flags that are applied to the wells c For fla
41. ate In this example rows F P are not in use layout i z z 7 g g 7 g z 7 7 z 5 3 EG A 7 3 g EJ a CC CJ CO DJ Ce Jen ee ee ee DJ DJ ee ee DJ DJ DJ DJ ee ee ee DJ Jen eee m ee ee Oe JJ OJ OJ JE OJ JO OJ OJ OJ GJ On On DJ OJ DJ ee ee DD DE DE DE DE De De Mice Mie Mie Mie Mee Mee Mee Dee Oe Mee Mee ee ee ee Dee Dee DSG EEE EE EE EEE EE ee EE EE EE EE ee oe Protein Thermal Shift Studies User Guide 69 4 Ligand Screening Studies Prepare the protein melt reactions Prepare the protein melt reactions Protein melt For consistency in Tm values we recommend that you keep the protein melt reactions reaction stability on ice until you are ready to load the instrument and start the run If the protein is thermally stable at ambient temperatures you may consider preparing the reactions in advance and leave the reaction plate at ambient temperature protected from light However the fluorescence levels will decrease over time and the Tm values will vary depending on the protein and its thermal stability If you want to prepare the reaction plates in advance we recommend that you first determine the benchtop stability of your protein melt reactions Required materials Required materials for protein melt reactions e Protein Thermal Shift Dye 1000X e Protein Thermal Shift Buffer e Water e Protein e Ligands or ligand titrations MicroAmp Optical Reaction Plate appropriate for your real t
42. ce Access the Help system by doing one of the following e Click in the toolbar of a study screen e Select Help gt Help Topics Protein Thermal Shift Studies User Guide 95 Documentation and Support Obtaining SDSs You can use the Help system to find topics of interest by Obtaining SDSs Reviewing the table of contents Searching for a specific topic Searching an alphabetized index Safety Data Sheets SDSs are available from www appliedbiosystems com sds Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtaining support For the latest services and support information for all locations go to www appliedbiosystems com At the website you can 96 Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches Protein Thermal Shift Studies User Guide analysis group analysis mode Boltzmann fit B fit Boltzmann Tm Tm B condition derivative Tm Tm D experiment file Glossary In Protein Thermal Shift studies one reference replicate gr
43. close the experiment file Note The melt curves in the real time PCR software may not exactly match the melt curves in the Protein Thermal Shift Software When the experiment files are imported into the Protein Thermal Shift Software the Protein Thermal Shift Software reduces the noise in the fluorescence data Example melt Melt curves for the buffer screening example file from the ViiA 7 System curves wus npc MM verget 1 Mi targes2 Bil teoet3 M Taroet 4 Set up the analysis This section provides instructions for setting up the protein thermal shift study using Protein Thermal Shift Software v1 0 For more information about how to use the software refer to the Protein Thermal Shift Software Help Setup guidelines The experiment files that you import into the study must contain analyzed melt curve data from a complete melt curve run e Set up the analysis group so that it contains experiment files from only one instrument TM fa Create and set up 1 In the Home screen of the Protein Thermal Shift Software click amp Create the study Study 2 Complete the Setup gt Properties screen e The Study Name cannot be more than 100 characters and cannot contain these characters V lt gt 1 The instrument selection must match the instrument type that you used to run the protein melt reactions and generate the experiment files 3 In the Setup gt Conditions screen define the c
44. creening study before performing a mutation screening study or a ligand screening study Replicates and For buffer screening studies we recommend that you prepare controls e At least 4 replicates of each reaction e Atleast 4 replicates of no protein controls NPCs Create and set up an experiment file for the instrument run This section contains general settings for creating and setting up an experiment file For detailed instructions see Instrument Page ViiA 7 Real Time PCR System page 12 StepOne and StepOnePlus Real Time PCR Systems page 14 7500 and 7500 Fast Real Time PCR Systems page 16 Ge file Setup Setting Experiment e Experiment type Melt Curve properties e Reagents Other e Ramp speed Fast or Standard Target e Reporter ROX properties e Quencher None Plate layout Assign targets to all wells in use Passive reference None Run method Reaction Volume Per Well 20 pL Thermal profile Step 1 Temp 25 C Time 2 minutes Step 2 Temp 99 C Time 2 minutes Ramp mode Continuous Ramp rate ViiA 7 System Step 1 1 6 C s Step 2 0 05 C s StepOne and StepOnePlus Systems and 7500 and 7500 Fast Systems 1 Optical Filters ViiA 7 System only Excitation Filter x4 580 10 Emission Filter m4 623 14 36 Protein Thermal Shift Studies User Guide Defining and assigning targets Example targets Example plate layout Chapt
45. d Reporter from the Plot dropdown list then select Target from the Color dropdown list 3 Review the melt curves e Do you see fluorescence signals in all of the sample wells No fluorescence signals in the sample wells may indicate missing dye or protein or an instrument problem e Do you see flat fluorescence levels in the NPC wells High fluorescence levels in the NPC wells may indicate protein contamination in the wells or protein melt reactions or it may indicate that the dye interacts with a component in the buffer Protein Thermal Shift Studies User Guide 71 4 Ligand Screening Studies Set up the analysis Do you see flat fluorescence levels in the LOC wells High fluorescence levels in the LOC wells but not in the NPC wells may indicate protein contamination in the ligand or ligand dye interactions Fluorescence from ligand dye interactions may mask the protein dye interactions e Do the replicates have similar melt curves 4 Save then close the experiment file Note The melt curves in the real time PCR software may not exactly match the melt curves in the Protein Thermal Shift Software When the experiment files are imported into the Protein Thermal Shift Software the Protein Thermal Shift Software reduces the noise in the fluorescence data Example melt Melt curves for the ligand screening example file from the ViiA 7 System curves o000 0 700000 Normalized Reporter Rn 0000 0 3000
46. e analysis settings Note The positive hits are determined according to the analysis settings that you specified page 23 For the example ligand titration study review the ATm Boltzmann positive hits In your own studies you may choose to review the ATm Derivative positive hits Note You must specify the reference replicate group to calculate ATm values and to determine positive hits 1 In the Analysis gt Replicate Results screen select the gt Plot by menu then select ATm Boltzmann 2 Select the Ne Show in Plot menu then select to show Positive Hits 3 Scan the Replicate Results Plot for positive hits The positive hits are replicate plot s within the green shaded area and labeled with the hit threshold in the bottom right corner of the plot Positive hit threshold from analysis settings Reference replicate plot ATm 0 C Positive hit Mean ATm exceeds the hit threshold Hit threshold gt 2 0 35 30 25 20 15 40 05 00 05 10 15 20 25 30 35 40 45 50 55 60 Temperature C j S Positive hit region Mean ATm exceeds the hit threshold 4 Review the ATm statistics for each replicate group in the Replicate Results Plot a Zoom in Click then click drag an area on the plot as many times as you need to magnify the plot Mh a h b Move the plot Click then click drag the plot until the replicate plot you want to review is in view 32 Protein Thermal Shift Studies User Guide
47. e before importing the experiment file into the Protein Thermal Shift Software IMPORTANT Keep the protein melt reactions on ice until you load the instrument 1 Ifnecessary transfer the experiment file that you created for the run to the computer that is connected to the instrument 2 In the Home screen of the Real Time PCR System Software click Open then select the experiment file you created for the run 3 Load the reaction plate into the instrument 4 In the Real Time PCR System Software click Run in the navigation pane then click START RUN Using the Real Time PCR System Software open the experiment file from the completed instrument run analyze and save the experiment file then review the melt curves Note You must analyze and save the experiment file in the Real Time PCR System Software before you can import it into the Protein Thermal Shift Software Some common troubleshooting causes are provided here For more detailed troubleshooting information see page 83 1 In the Home screen of the Real Time PCR System Software click Open then select the experiment file from the instrument run 2 View the melt curves Real time PCR System Software View the melt curve ViiA 7 Software Click Analysis gt Melt Curve Plot in the navigation pane select Normalized Reporter from the Plot dropdown list then select Target from the Color dropdown list StepOne Software or Click Analysis gt
48. e to reanalyze using the Auto Multiple Tm analysis mode d Review the number of melt phases in the derivative melt curves e Do all replicate groups have the same number of melt phases e For each replicate group are there outliers with a different number of melt phases than the other samples in the replicate group You may consider omitting outliers from analysis Review the regions Review the ROAs detected by the software If necessary edit the ROAs of analysis ROA Note If no melt phases are detected no ROAs are defined Negative controls should have no melt phases and no ROAs 1 In the Analysis gt Well Results screen confirm that each ROA meets the following criteria e For melt curves with one melt phase the curve within the ROA resembles a sigmoidal profile e At the start temperature the signal is relatively flat e Atthe end temperature the signal has already reached its maximum 2 For each replicate group edit the ROAs so that all of the wells in the replicate group have the same ROAs a Select the replicates click Define ROA in the toolbar above the melt curve plots then click drag an area in one of the plots to define a melt phase and replace the ROA Repeat for each melt phase you identify b If necessary adjust the start and end temperatures for each ROA To move the ROA Starting from within the ROA click drag the ROA e To adjust the start and end temperatures individually Click drag the
49. ed content in the reaction plate The number of rows and columns in the grid corresponds to the instrument reaction block that you use In the Protein Thermal Shift Software you can use the plate layout as a selection tool to make or view condition assignments or to omit wells from analysis The plate layout can be saved as a jpg or a png image file In Protein Thermal Shift studies a sample replicate group with an average ATm value that exceeds the threshold set in the analysis settings for the study A positive hit can identify conditions for example buffer ligand or mutation that increase or decrease protein thermal stability relative to the reference replicate group Protein Thermal Shift Studies User Guide reference region of analysis replicate group ROA study task Tm Tm B Tm D Glossary In Protein Thermal Shift studies the task assigned to one replicate group within an analysis group The results from the reference replicate group are used to calculate ATm values for sample replicate groups A range of temperatures in the melt curves from which the fluorescence data are used to calculate the Tm The region of analysis can be detected by the software auto analysis mode or manually defined by the user manual analysis mode Up to 6 regions of analysis can be defined for a well In Protein Thermal Shift studies a set of identical reactions with the same set of conditions the same task
50. eference replicate group results Lower 95 confidence limit Upper 95 confidence limit Median Mean Single Tm Analysis Sample replicate group results Lower 95 confidence limit Upper 95 confidence limit Median Mean Multiple Tm Analysis Sample replicate group results one plot for each ROA af d Diamond color is the task color Reterence Sample replicate group replicate group Datapoint color is the experiment file color Experimentt eds Experiment2 eds Datapoint shape indicates omit status X Omitted well Included well Protein Thermal Shift Studies User Guide 29 Getting Started with a Protein Thermal Shift Study Review the replicate results Review the In the Analysis gt Replicate Results screen review the Boltzmann Tm statistics to Boltzmann Tm evaluate the variability among replicates The Boltzmann Tm statistics are plotted statistics along the x axis for each replicate group For the example ligand titration study we provide instructions for reviewing the Boltzmann Tm statistics For your own studies you may also review derivative Tm statistics 1 In the Analysis gt Replicate Results screen select lt gt Plot by then select Tm Boltzmann Note Select Tm Derivative to review the derivative Tm statistics 2 Specify the condition hierarchy to group the replicate plots and change the order of conditions in the plot Note Changing the condition hie
51. ene eames te 95 Related documentation 0 020 244 gcd ee le sake bay a era 95 Obtaining information from the Help system 202 cece cette eee 95 Obtaining SDSS areas Le de 96 Obtaining support inserat ganne a poet he Ageless adelens je Ben ea eases aa Ute 96 Glossary 46 05 52c nie sleder eee 97 Protein Thermal Shift Studies User Guide About This Guide IMPORTANT Before using this product read and understand the information the Safety appendix in this document Purpose This guide is designed to help you quickly learn how to perform Protein Thermal Shift studies using Protein Thermal Shift reagents Applied Biosystems Real Time PCR Systems and Protein Thermal Shift Software v1 0 This guide provides step by step procedures on e How to perform an example ligand titration study using the Protein Thermal Shift Starter Kit and the example experiment files installed with the Protein Thermal Shift Software e How to perform and troubleshoot your buffer screening mutation screening and ligand screening studies using Protein Thermal Shift reagents and Protein Thermal Shift Software v1 0 User attention words Five user attention words may appear in this document Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information
52. er 2 Buffer Screening Studies Create and set up an experiment file for the instrument run We recommend that you define and assign targets so you can review the melt curves for the replicate groups in the Real Time PCR System Software before importing the experiment files into the Protein Thermal Shift study Define the following targets e One target for each buffer condition e Define a target for the no protein control NPC IMPORTANT Make sure that the well assignments correspond exactly with the arrangement of reactions in the reaction plate Setup errors may result in an incorrect grouping of replicates In the ViiA 7 System buffer screening example file targets were defined for each buffer and the no protein control NPC Target Name Reporter Quencher Color Target 1 Rox v None v v In this example columns 9 24 and rows K P are not in use DE DE DE DE DE OE u ee Om OG 06 ee 068 Om u DE Om ee 06 OG 068 Om u DE 068 68 068 068 68 068 me DE 068 68 068 068 68 068 Des De De Os Dee O59 D69 DE De De De Oe Cee Oe O69 DE ME EE EE HE DE 08 EE EE GE Ee OF Protein Thermal Shift Studies User Guide 37 2 Buffer Screening Studies Prepare the protein melt reactions Prepare the protein melt reactions Protein melt For consistency in Tm values we recommend that you keep the protein melt reactions reaction stability on ice until you are ready to load the instrument and start
53. erize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory e Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations e IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Protein Thermal Shift Studies User Guide 93 Safety Biological hazard safety Biological hazard safety 94 AN AN WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or
54. ettings and analyze e Click Apply to apply the analysis settings and reanalyze while keeping the Analysis Settings dialog box open or e Click OK to apply the analysis settings reanalyze and close the Analysis Settings Protein Thermal Shift Studies User Guide 57 3 Mutation Screening Studies Review the well results Review the well results Review the melt curves 58 Using the Protein Thermal Shift Software review the melt curves and well table and optimize the analysis in the Analysis gt Well Results screen The Well Results screen displays fluorescence and derivative melt curve plots calculated Tm values individual well results and flags As necessary edit the analysis settings edit the baseline edit the region of analysis edit the analysis mode and omit outliers This section provides guidance on how to review and interpret the well results Some common troubleshooting causes are provided For more detailed troubleshooting information see page 83 Review the melt curve plots to visualize the fluorescence and derivative fluorescence data If necessary change the analysis mode Note For NPC wells the derivative melt curves are not displayed 1 Inthe Analysis gt Well Results screen select Ne Show in Plot then select to show these plot components e Unselected Wells e Legend 2 Select a Color By then select Protein to color the melt curves according to the protein condition value assigned for
55. g study to identify the optimal buffer pH or salt concentration for storing a protein Buffer screening may also be used to identify a buffer system for a protein that aggregates or precipitates from solution or to improve protein crystal formation for x ray crystallography Perform a buffer screening study Create and set up an experiment file for the instrument run page 36 l Prepare the protein melt reactions page 38 l Run the protein melt reactions page 39 l l Review the well results page 42 l Review the replicate results page 46 Set up the analysis page 40 Example experiment files and plate template files are located in the examples folder lt drive gt Program Files Applied Biosystems Protein Thermal Shift Software examples where lt drive gt is where you installed the software To view the data for the buffer screening example used in this chapter use the Buffer Screening Example ViiA7 eds and Buffer Screening Example Setup ViiA7 csv files in the ViiA7 Example Files folder Protein Thermal Shift Studies User Guide 35 2 Buffer Screening Studies General guidelines General guidelines For general guidelines that apply to all Protein Thermal Shift studies see page 9 Experimental With Protein Thermal Shift studies you can perform a series of buffer screening design studies to identify the optimal buffer pH and salt concentration for a protein You may also perform a buffer s
56. ge 14 7500 and 7500 Fast Real Time PCR Systems page 16 a file Setup Setting Experiment e Experiment type Melt Curve properties e Reagents Other e Ramp speed Fast or Standard Target e Reporter ROX properties e Quencher None Plate layout Assign targets to all wells in use Passive reference None Run method Reaction Volume Per Well 20 pL Thermal profile Step 1 Temp 25 C Time 2 minutes Step 2 Temp 99 C Time 2 minutes Ramp mode Continuous Ramp rate ViiA 7 System Step 1 1 6 C s Step 2 0 05 C s StepOne and StepOnePlus Systems and 7500 and 7500 Fast Systems 1 Optical Filters ViiA 7 System only Excitation Filter x4 580 10 Emission Filter m4 623 14 52 Protein Thermal Shift Studies User Guide Defining and assigning targets Example targets Example plate layout Chapter 3 Mutation Screening Studies Create and set up an experiment file for the instrument run We recommend that you define and assign targets so you can review the melt curves for the replicate groups in the Real Time PCR System Software before importing the experiment files into the Protein Thermal Shift study Define the following targets One target for each protein mutation e Define a target for the no protein control NPC IMPORTANT Make sure that the well assignments correspond exactly with the arrangement of reactions in the reaction plate Setup errors may result in a
57. gged wells select the well in the Well Table then review the melt curves for the well compared to the other wells in the replicate group d Omit wells from analysis as necessary 3 For each replicate group select all the wells in the replicate group then review the Tm B Boltzmann Tm and the Tm D derivative Tm in the Well Table and in the melt curves Note In the melt curves the Boltzmann Tm is a green dashed vertical line and the derivative Tm is black dotted vertical line Are the Tm B or Tm D values significantly different from the Tm values for other wells in the replicate group Do any replicates have melt curves that are inconsistent with the other melt curves for the replicate group If so you may consider omitting wells from analysis e Are the melt curves within the replicate group similar 4 If you omitted any wells from the analysis click gt Analyze Review the 1 In the Analysis gt Well Results screen select Le Show in Plot then select to show Boltzmann fit these plot components e Boltzmann Fit e Unselected Wells e Legend 2 Scroll through each well in the Well Table then compare the fluorescence melt curve to the Boltzmann fit curve dark green thick curve and review the value in the B Fit Boltzmann Fit column of the Well Table e How well does the fluorescence melt curve correspond to the Boltzmann fit curve e Js the B Fit value close to 1 e Is the B Fit value similar among wells in
58. he ViiA 7 System example buffer screening study observe the following results For the ViiA 7 System example mutation screening study observe the following e Fluorescence levels are flat in the NPC wells e For the sample and reference replicate groups the fluorescence levels as displayed in the fluorescence melt curve are not significantly different so you do not need to edit the baseline e The sample and reference wells contain one peak in the derivative melt curve e Within each replicate group The fluorescence melt curves and the Boltzmann Tm values are similar The derivative melt curves and the derivative Tm values are similar and there are no outliers e For each well the region of analysis meets the recommended criteria Protein Thermal Shift Studies User Guide 61 3 Mutation Screening Studies Review the replicate results Review the replicate results Using the Protein Thermal Shift Software review the Tm statistics for the replicate groups and look for positive hits in the Analysis gt Replicate Results screen This section provides guidance on how to review and interpret the replicate results Some common troubleshooting causes are provided For more detailed information on troubleshooting see page 83 Review the Tm In the Analysis gt Replicate Results screen review the Tm statistics to evaluate the statistics variability among replicates The Tm statistics for each ROA are plotted along the x
59. hold gt 20 35 30 25 20 15 40 05 00 05 10 15 20 25 30 35 40 45 50 55 60 Temperature C _ gt _ Positive hit region Mean ATm exceeds the hit threshold 4 Review the ATm statistics for each replicate group in the Replicate Results Plot a Zoom in Click then click drag an area on the plot as many times as you need to magnify the plot 1 ath b Move the plot Click then click drag the plot until the replicate plot you want to review is in view 48 Protein Thermal Shift Studies User Guide Chapter 2 Buffer Screening Studies Review the replicate results c Place the cursor over the replicate plot then wait to view a tooltip with the ATm statistics for the replicate group Note Click 2 to restore the default zoom 5 In the Replicate Groups table review the positive hits 4 in the Hits B or Hits D column and review the ATm B or ATm D statistics in the table Protein Thermal Shift Studies User Guide 49 2 Buffer Screening Studies Review the replicate results 50 Protein Thermal Shift Studies User Guide Mutation Screening Studies Perform a mutation screening study to screen for mutations that increase or decrease the thermal stability of a protein Perform a mutation screening study Create and set up an experiment file for the instrument run page 52 l Prepare the protein melt reactions page 54 l Run the protein melt reactions page 55 l l Review
60. ift Software IMPORTANT Keep the protein melt reactions on ice until you load the instrument 1 Ifnecessary transfer the experiment file that you created for the run to the computer that is connected to the instrument 2 In the Home screen of the Real Time PCR System Software click Open then select the experiment file you created for the run 3 Load the reaction plate into the instrument 4 In the Real Time PCR System Software click Run in the navigation pane then click START RUN Using the Real Time PCR System Software open the experiment file from the completed instrument run analyze and save the experiment file then review the melt curves Note You must analyze and save the experiment file in the Real Time PCR System Software before you can import it into the Protein Thermal Shift Software Some common troubleshooting causes are provided here For more detailed troubleshooting information see page 83 1 In the Home screen of the Real Time PCR System Software click Open then select the experiment file from the instrument run 2 View the melt curves Real time PCR System Software View the melt curve ViiA 7 Software Click Analysis gt Melt Curve Plot in the navigation pane select Normalized Reporter from the Plot dropdown list then select Target from the Color dropdown list StepOne Software or Click Analysis gt Melt Curve in the navigation pane select 7500 Software Normalize
61. ift studies Item Catalog no Protein Thermal Shift Starter Kit 4462263 Protein Thermal Shift Dye Kit 4461146 Protein Thermal Shift Software v1 0 Additional License 4466038 Protein Thermal Shift Software v1 0 10 licenses 4466037 Protein Thermal Shift Studies User Documentation Set 4463373 Table 2 Applied Biosystems Real Time PCR Systems Item Catalog no Applied Biosystems ViiA 7 Real Time PCR System e ViiA 7 Real Time PCR System with 384 Well Block e 4453536 e ViiA 7 Real Time PCR System with 96 Well Fast Block e 4453535 e ViiA 7 Real Time PCR System with 96 Well Block e 4453534 Applied Biosystems StepOnePlus Real Time PCR System e StepOnePlus Real Time PCR System e 4376600 e StepOnePlus Real Time PCR System with Laptop e 4376598 e StepOnePlus Real Time PCR System with Tower e 4376599 Applied Biosystems StepOne Real Time PCR System StepOne Real Time PCR System 4376357 StepOne Real Time PCR System with Laptop 4376373 e StepOne Real Time PCR System with Tower 4376374 Applied Biosystems 7500 Fast Real Time PCR System e 7500 Fast Real Time PCR System with Dell Notebook e 4351106 e 7500 Fast Real Time PCR System with Dell Tower e 4351107 Applied Biosystems 7500 Real Time PCR System e 7500 Real Time PCR System with Dell Notebook e 4351104 e 7500 Real Time PCR System with Dell Tower e 4351105 Protein Therma
62. ile If you observe that the Tm is close to the beginning or close to the end of the melt curve adjust the temperature range so that the Tm is in the middle of the temperature range and you are able to observe a complete protein melt Because of possible plate to plate variability we recommend that you set up the plates so that each plate contains a reference group and an analysis group does not span multiple plates For all Protein Thermal Shift studies we recommend that you prepare at least 4 replicates of each protein melt reaction to ensure statistically significant results For Protein Thermal Shift studies we recommend that you prepare controls e No Protein Control NPC Protein melt reactions that contain only buffer water and dye NPC wells with high fluorescence signals and melt phases may indicate contamination in wells or protein melt reactions Ligand Only Control LOC Protein melt reactions that contain only ligand buffer water and dye LOC wells with melt profiles distinct from NPC wells may indicate ligand dye interactions Ligands that bind the dye and affect the fluorescence levels may mask the presence or absence of protein dye interactions Protein Thermal Shift Studies User Guide Supported real time PCR systems Real time PCR system maintenance schedule Chapter 1 Getting Started with a Protein Thermal Shift Study 1 General guidelines Melt curve experiment files from the following App
63. ime PCR instrument e MicroAmp Optical Adhesive Film appropriate for your reaction plate Prepare the protein We recommend that you prepare four replicates of each reaction melt reactions 1 Prepare a fresh dilution of Protein Thermal Shift Dye 1000X to 8X 2 Place the appropriate reaction plate or tubes on ice then prepare the protein melt reactions e Make sure that the arrangement of reactions in the reaction plate corresponds exactly with the well assignments in the experiment file IMPORTANT Setup errors may result in an incorrect grouping of the data and incorrect Tm statistics e Add reaction components to the plate in the order listed Component Volume Protein Thermal Shift Buffer 5 0 uL Water protein ligand 12 5 uL Diluted Protein Thermal Shift Dye 8X 2 5 uL Total volume for each control reaction 20 0 pL 3 Pipet each reaction up and down 10 times to mix well 4 Seal the plate with MicroAmp Optical Adhesive Film spin it at 1000 rpm for 1 minute then place it on ice 70 Protein Thermal Shift Studies User Guide Chapter 4 Ligand Screening Studies Run the protein melt reactions Run the protein melt reactions Load and run the reactions Review the melt curves Load and run the protein melt reactions on a supported Applied Biosystems Real Time PCR System then analyze and save the experiment file before importing the experiment file into the Protein Thermal Sh
64. in combination with software or products not supplied or authorized by Life Technologies and modification or repair of the product not authorized by Life Technologies The foregoing provisions set forth Life Technologies sole and exclusive representations warranties and obligations with respect to its products and Life Technologies makes no other warranty of any kind whatsoever expressed or implied including without limitation warranties of merchantability and fitness for a particular purpose whether arising from a statute or otherwise in law or from a course of dealing or usage of trade all of which are expressly disclaimed The remedies provided herein are the buyer s sole and exclusive remedies Without limiting the generality of the foregoing in no event shall Life Technologies be liable whether in contract tort warranty or under any statute including without limitation any trade practice unfair competition or other statute of similar import or on any other basis for direct indirect punitive incidental multiple consequential or special damages sustained by the buyer or any other person or entity whether or not foreseeable and whether or not Life Technologies is advised of the possibility of such damages including without limitation damages arising from or related to loss of use loss of data failure or interruption in the operation of any equipment or software delay in repair or replacement or for loss of revenue or p
65. indicate a positive hit On the Flags tab specify settings for applying flags a Select the flags to use in the analysis b For the High Background High NPC Low Signal and Poor Fit flags specify the condition and threshold for applying the flag Apply the analysis settings and analyze e Click Apply to apply the analysis settings and reanalyze while keeping the Analysis Settings dialog box open or e Click OK to apply the analysis settings reanalyze and close the Analysis Settings Protein Thermal Shift Studies User Guide 73 4 Ligand Screening Studies Review the well results Review the well results Using the Protein Thermal Shift Software review the melt curves and well table and optimize the analysis in the Analysis gt Well Results screen The Well Results screen displays fluorescence and derivative melt curve plots calculated Tm values individual well results and flags As necessary edit the analysis settings edit the baseline edit the region of analysis edit the analysis mode and omit outliers This section provides guidance on how to review and interpret the well results Some common troubleshooting causes are provided For more detailed troubleshooting information see page 83 Review the melt Review the melt curve plots to visualize the fluorescence and derivative fluorescence curves data If necessary change the analysis mode Note For NPC wells the derivative melt curves are not displayed 1
66. itions select the wells with those conditions select the Assign checkbox for the target then select U for the Task c Optional For no protein controls NPC select the NPC wells select the Assign checkbox for the NPC target then select N for the Task d For the passive reference select None 6 Make sure that the well assignments correspond exactly with the arrangement of reactions in the reaction plate IMPORTANT Setup errors may result in an incorrect grouping of replicates 7 Complete the Run Method screen to define the melt curve a For the Reaction Volume Per Well enter 20 uL b For the ramp mode select Continuous c Define the thermal profile Time o Step Ramp rate Temp C ne 1 100 25 0 02 00 2 1 99 0 02 00 Protein Thermal Shift Studies User Guide 17 Getting Started with a Protein Thermal Shift Study Prepare the protein melt reactions Melt Curve Stage Continuous StepAndHold Step 1 Step 2 Select File gt Save then enter a file name and select a location for the experiment file Prepare the protein melt reactions About the example For the example ligand titration study prepare 5 sets of protein melt reactions using study protein melt the control protein and control ligand in the Protein Thermal Shift Starter Kit reactions Control protein and 0 0 mM control ligand Control protein and 0 1 mM control ligand Control protein and
67. l Shift Studies User Guide 91 Ordering Information 92 Table 3 Plates and accessories for 384 well systems Item Catalog no Applied Biosystems MicroAmp Optical Adhesive Film e 100 films e 4311971 e 25 films e 4360954 Applied Biosystems MicroAmp Optical 384 Well Reaction Plate 4343370 1000 plates Applied Biosystems MicroAmp Optical 384 Well Reaction Plate with Barcode e 1000 plates e 4343814 e 500 plates e 4326270 e 50 plates e 4309849 Table 4 Plates and accessories for 96 well systems Item Catalog no Applied Biosystems MicroAmp Optical Adhesive Film e 100 films e 4311971 e 25 films e 4360954 Applied Biosystems MicroAmp Fast Optical 96 Well Reaction Plate with Barcode 0 1 mL e 200 plates e 4366932 e 20 plates e 4346906 Table 5 Plates and accessories for 48 well systems Item Catalog no Applied Biosystems MicroAmp 48 Well Optical Adhesive Film e 100 films e 4375323 e 25 films e 4375928 Applied Biosystems MicroAmp Fast Optical 48 Well Reaction Plate 4375816 20 plates Protein Thermal Shift Studies User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before
68. le or reference wells do you observe flat melt curves If so condition assignments may be incorrect or a component is missing from the protein melt reactions Within each replicate group are the fluorescence melt curves similar to each other Within each replicate group are the derivative melt curves similar to each other If the melt curves for the replicates are dissimilar pipetting errors may have occurred during reaction setup or condition assignments may be incorrect In the Protein Thermal Shift Software the analysis mode is the method for defining the regions of analysis ROA and determining the derivative Tm for a melt curve The analysis mode is displayed in the Analysis Mode column of the Well Table in the Analysis screens Analysis mode Description Auto Single Tm The software detects one melt phase defines one ROA and determines one derivative Tm within the ROA By default the melt curves are analyzed using the Auto Single Tm analysis mode Auto Multiple Tm The software detects more than one melt phase defines an ROA for each melt phase and determines one derivative Tm for each ROA The software can detect up to six ROAs for each melt curve If the derivative melt curve shows multiple melt phases change the analysis mode to Auto Multiple Tm Manual You define the ROA within the software then the software determines one derivative Tm for each ROA that you defined You can define up to
69. lied Biosystems Real Time PCR Systems and system software are supported Applied Biosystems Real Time PCR System System software versions ViiA 7 Real Time PCR System ViiA 7 Software v1 0 and v1 1 StepOne and StepOnePlus Real Time PCR Systems StepOne Software v2 1 and v2 2 7500 and 7500 Fast Real Time PCR Systems 7500 Software v2 0 4 and v2 0 5 Note Other system software versions for the real time PCR systems listed may be accepted by the Protein Thermal Shift Software By default a warning message is displayed when you import an experiment file from a system software version that is not supported Perform routine maintenance of your Applied Biosystems real time PCR system to ensure proper operation Refer to the appropriate user guide for your real time PCR system for the maintenance schedule and detailed instructions Maintenance Task type Instrument Clean the surface of the instrument with a lint free cloth Computer e Check the computer disk space and archive or back up your experiment files e Power off the computer controlling the instrument then after 30 seconds power on the computer e Defragment the computer hard drive Calibrations e Perform a background calibration e Perform a spatial calibration e Perform a dye calibration with ROX dye Protein Thermal Shift Studies User Guide 11 Getting Started with a Protein Thermal Shift Study Create and set up an experimen
70. m and maximum Tm values for the replicate group Is the range of Tm values for the replicate group within 1 degree If the range of Tm values exceeds 1 degree review the data for each replicate 6 Omit outliers as necessary then click gt Analyze Protein Thermal Shift Studies User Guide 79 4 Ligand Screening Studies Review the replicate results Review the positive In the Analysis gt Replicate Results screen review the positive hits to identify the hits conditions that produce the maximum effect on thermal stability Replicate groups with positive hits have ATm values that exceed the threshold set in the analysis settings Note The positive hits are determined according to the analysis settings that you specified Note You must specify the reference replicate group to calculate ATm values and to determine positive hits 1 In the Analysis gt Replicate Results screen select lt gt Plot by then select the type of ATm statistics to review e ATm Boltzmann e ATm Derivative 2 Select the _ Show in Plot menu then select to show Positive Hits 3 Scan the green shaded area of the Replicate Results Plot for positive hits Positive hit threshold from analysis settings l Reference replicate plot ATm 0 C Positive hit Mean ATm exceeds the hit threshold Hit threshold gt 20 35 30 25 20 15 40 05 00 05 10 15 20 25 30 35 40 45 50 55 60 Temperature C _ gt _ Positive hit region Mean ATm
71. mal Shift Control Protein Purpose of the dye kit Dye kit contents and storage Part number After you learn how to perform a Protein Thermal Shift study you can use the Protein Thermal Shift Dye Kit The Protein Thermal Shift Dye Kit contains buffer and dye to perform the Protein Thermal Shift reactions with your proteins of interest The Protein Thermal Shift Dye Kit contains one box Quantity Storage conditions Component 4461146 Sufficient for 2000 reactions Protein Thermal Shift Buffer Protein Thermal Shift Dye Room temperature RT 18 C to 25 C Protein Thermal Shift Studies User Guide Chapter 1 Getting Started with a Protein Thermal Shift Study General guidelines Materials required For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies but not included contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions Item Source Applied Biosystems Real Time PCR System Life Technologies Applied Biosystems Protein Thermal Shift Software v1 0 Life Technologies Applied Biosystems MicroAmp Optical Reaction Plates Life Technologies Applied Biosystems MicroAmp Optical Adhesive Film Life Technologies Centrifuge with plate adapters Major laboratory suppliers MLS Microce
72. mal Shift Dye 8X 2 5 uL Total volume for each control reaction 20 0 pL 3 Pipet each reaction up and down 10 times to mix well 4 Seal the plate with MicroAmp Optical Adhesive Film spin it at 1000 rpm for 1 minute then place it on ice Run the protein melt reactions Load and run the reactions Load and run the protein melt reactions on a supported Applied Biosystems Real Time PCR System then analyze and save the experiment file before importing the experiment file into the Protein Thermal Shift Software IMPORTANT Keep the protein melt reactions on ice until you load the instrument Refer to your instrument user guide for detailed instructions on how to transfer experiment files to the instrument computer or instrument how to operate the instrument how to start the run from an instrument touchscreen and how to monitor the run 1 Ifnecessary transfer the experiment file that you created for the run to the computer that is connected to the instrument 2 In the Home screen of the Real Time PCR System Software click Open then select the experiment file you created for the run 3 Load the reaction plate into the instrument 4 In the Real Time PCR System Software click Run in the navigation pane then click START RUN Protein Thermal Shift Studies User Guide 19 Getting Started with a Protein Thermal Shift Study Run the protein melt reactions Review the melt curves 20 Using the
73. n incorrect grouping of replicates In the ViiA 7 System mutation screening example file targets were defined for each protein and the no protein control NPC Target Name Reporter Quencher Color Wild Type Rox vw None v v In this example columns 13 24 and rows I P are not in use 1 3 4 5 6 7 38 9 10 11 12 DO ee ee ee eee eee eee eee eee ee DO DO ee ee ee ee ee eee eee eee Protein Thermal Shift Studies User Guide 53 3 Mutation Screening Studies Prepare the protein melt reactions Prepare the protein melt reactions Protein melt For consistency in Tm values we recommend that you keep the protein melt reactions reaction stability on ice until you are ready to load the instrument and start the run If the protein is thermally stable at ambient temperatures you may consider preparing the reactions in advance and leave the reaction plate at ambient temperature protected from light However the fluorescence levels will decrease over time and the Tm values will vary depending on the protein and its thermal stability If you want to prepare the reaction plates in advance we recommend that you first determine the benchtop stability of your protein melt reactions Required materials Required materials for protein melt reactions Protein Thermal Shift Dye 1000X Protein Thermal Shift Buffer Water Protein sample
74. ndition Note For the example buffer screening study set up the hierarchy so that Buffer is at the bottom in the dialog box and displayed on the far left side of the Replicate Results Plot Scan the Replicate Results Plot to review the conditions that affect the Tm values relative to the reference replicate group Reference replicate plot red Sample replicate plot blue Sample replicate plot blue Protein Thermal Shift Studies User Guide Chapter 2 Buffer Screening Studies Review the replicate results 4 Review the Tm statistics for each replicate group in the Replicate Results Plot a Zoom in Click then click drag an area on the plot one or more times am b Move the plot Click gt then click drag the plot until the replicate plot of interest is in view c Place the cursor within the diamond then wait to view a tooltip with the Tm statistics for the replicate group Tm Statistics Mean 43 75 Lower 95 43 67 Upper 95 43 83 Median 43 77 Std Error 0 04 Min 42 96 Max 43 90 d To examine outliers place the cursor over a datapoint then wait to view a tooltip with the well information experiment file name and the Tm selected for the plot Well B21 Experiment Ligand_Screening_Example_ViiA7 eds Tm Boltzmann 43 35 Note Click I to restore the default zoom 5 In the Replicate Groups table review the Tm statistics for each replicate group e Std
75. ntrifuge MLS Microcentrifuge tubes MLS Pipettors and pipette tips MLS Vortexer MLS Water MLS General guidelines Protein Before you perform a Protein Thermal Shift study first consider whether the protein considerations of interest is a suitable candidate for this method e Is the protein thermally stable as determined by other methods If the Tm for the protein is greater than 98 C the protein melt reactions will produce melt curves with no distinct melt phase You may observe flat fluorescence signals a decrease in signal or high signals depending on the protein You may need to use other methods to screen for conditions that increase thermal stability of the protein For thermally stable proteins you may consider performing Protein Thermal Shift studies to screen for buffers ligands or mutations that decrease thermal stability of the protein e In the native state of a protein are there external hydrophobic sites If so you may observe a high initial background signal Conversely if the protein does not contain sufficient hydrophobic residues you may observe low fluorescence signals e Does the protein of interest comprise more than one domain or form oligomers If so you may observe a multi state model of unfolding and multiple melt phases in the melt curves For proteins that comprise more than one domain you may consider separate expression of just the domain with the active site For proteins tha
76. o use the software and refund the buyer s purchase price for the software If there is a defect in the media covered by the above warranty and the media is returned to Life Technologies within the ninety 90 day warranty period Life Technologies will replace the defective media Life Technologies does not warrant that the software will meet buyer s requirements or conform exactly to its documentation or that operation of the software will be uninterrupted or error free Any applicable warranty period under these sections begins on the earlier of the date of installation or ninety 90 days from the date of shipment for software installed by Life Technologies personnel For all software installed by the buyer or anyone other than Life Technologies the applicable warranty period begins the date the software is delivered to the buyer Protein Thermal Shift Studies User Guide 89 El Software Warranty Information Limited product warranty Warranty claims Warranty exceptions Warranty limitations 90 Warranty claims must be made within the applicable warranty period The above warranties do not apply to defects resulting from misuse neglect or accident including without limitation operation outside of the environmental or use specifications or not in conformance with the instructions for the instrument system software or accessories improper or inadequate maintenance by the user installation of software or interfacing or use
77. oftware For more information about how to use the software refer to the Protein Thermal Shift Software Help Select Start gt All Programs gt Applied Biosystems gt Protein Thermal Shift Software v1 0 F 1 In the Home screen click Create Study in the toolbar 2 In the Setup gt Properties screen enter a unique name for the study Note The Study Name cannot be more than 100 characters and cannot contain these characters lt gt 1 3 For the Instrument select the instrument that you used to run the protein melt reactions IMPORTANT The instrument selection must match the instrument type that you used to run the protein melt reactions and generate the experiment files Guidelines for the experiment files that you import into the study The experiment file must contain analyzed and saved melt curve data from a complete melt curve run The instrument type in the experiment file must match the instrument type selected for the study Note The data needed for the study are extracted from the eds file The Protein Thermal Shift Software does not create modify save or export eds files Protein Thermal Shift Studies User Guide 21 Getting Started with a Protein Thermal Shift Study Set up the analysis Set up the example ligand titration study About the analysis settings 22 First import the example experiment file eds then set up the plate using the example plate templa
78. onditions and condition values then list the analysis groups for your study 40 Protein Thermal Shift Studies User Guide Review the analysis settings 8 Chapter 2 Buffer Screening Studies 2 Set up the analysis In the Setup gt Experiment Files screen click Import then select the experiment file eds for the instrument type that you selected for the study Repeat for each experiment file to import into the study Note The experiment file name and data are imported Plate setup information is not imported For each experiment file assign the conditions task and analysis group to the wells that contained protein melt reactions Make sure that the condition assignments correspond exactly with the contents of the reaction plate IMPORTANT Setup errors may result in an incorrect grouping of the data and incorrect Tm statistics For each analysis group assign the reference replicate group a Select the wells for the replicate group to use as the reference b Click M Assign then select Reference as the Task Click Ca Save in the toolbar to save and analyze the study Review and edit the analysis settings to optimize the analysis for your study For the examples shown in this user guide no changes were made to the analysis settings If you are reviewing the data in the example studies provided with the software try revising the analysis settings to see how the settings affect the positive hits and the flag
79. or buffers that affect the thermal stability of the protein of interest Protein Thermal Shift Studies Mutation 4461811 Provides brief step by step procedures for performing Screening Quick Reference Protein Thermal Shift studies to screen for mutations that affect the thermal stability of the protein of interest Protein Thermal Shift Software Help 4461805 Provides detailed instructions for using the software to manage studies set up the analysis analyze and review the data and to publish the data Also contains video demos on how to set up the plate Applied Biosystems ViiA 7 Real Time 4442661 Explains how to calibrate maintain network and secure the PCR System User Guide Calibration ViiA 7 System Maintenance Networking and Security Applied Biosystems ViiA 7 Real Time 4441434 Explains how to perform experiments on the ViiA 7 System PCR System Getting Started Guide Applied Biosystems StepOne and 4376782 Explains how to install network and maintain the StepOne StepOnePlus Real Time PCR Systems and StepOnePlus Systems Installation Networking and Maintenance Guide Applied Biosystems 7500 7500 Fast 4387777 Explains how to install and maintain the 7500 and 7500 Fast Real Time PCR Systems System Maintenance Guide Systems Obtaining information from the Help system The Protein Thermal Shift Software has a Help system that describes how to use each feature of the user interfa
80. or positive hits in the Analysis gt Replicate Results screen This section provides guidance on how to review and interpret the replicate results Some common troubleshooting causes are provided For more detailed information on troubleshooting see page 83 In the Analysis gt Replicate Results screen review the Tm statistics to evaluate the variability among replicates The Tm statistics for each ROA are plotted along the x axis for each replicate group 1 In the Analysis gt Replicate Results screen select lt gt Plot by then select the type of Tm statistics to review e Tm Boltzmann e Tm Derivative Specify the condition hierarchy to group the replicate plots and change the order of conditions in the plot Note Changing the condition hierarchy does not affect the results it only changes how the replicate plots are grouped and the order in which the conditions are displayed in the Replicate Results Plot a Click Condition Hierarchy above the top right corner of the plot b In the dialog box select a condition then use the Up and Down arrows to change the hierarchy of conditions The condition at the top most level of the hierarchy is displayed on the far right side of the Replicate Results Plot and the replicate plots are grouped according to the top most condition Note For the example ligand screening study set up the hierarchy so that Ligand is at the bottom in the dialog box and displayed on the far lef
81. ou see flat fluorescence levels in the NPC wells High fluorescence levels in the NPC wells may indicate protein contamination in the wells or protein melt reactions or it may indicate that the dye interacts with a component in the buffer e Do you see flat fluorescence levels in the LOC wells With the Protein Thermal Shift Control Ligand high fluorescence levels in the LOC wells but not in the NPC wells may indicate protein contamination in the control ligand Protein Thermal Shift Studies User Guide Chapter 1 Getting Started with a Protein Thermal Shift Study 1 Set up the analysis With your own samples high fluorescence levels in the LOC wells but not in the NPC wells may indicate protein contamination in the control ligand or ligand dye interactions e Do the replicates have similar melt curves 4 Save then close the experiment file Note The melt curves in the real time PCR software may not exactly match the melt curves in the Protein Thermal Shift Software When the experiment files are imported into the Protein Thermal Shift Software the Protein Thermal Shift Software reduces the noise in the fluorescence data Set up the analysis Start the analysis software Create a study Experiment file guidelines This section provides instructions for setting up the protein thermal shift study using Protein Thermal Shift Software v1 0 and the example ligand screening files that are installed with the s
82. oup and multiple sample replicate groups analyzed together to calculate ATm values and to determine positive hits In the Protein Thermal Shift Software the method of defining the regions of analysis ROAs e Auto The software defines the ROA s Auto Single Tm The software detects one ROA and determines one Tm within the ROA Auto Multiple Tm The software detects more than one ROA and determines one Tm for each ROA e Manual Each ROA is manually defined within the Protein Thermal Shift Software The analysis mode is displayed in the Analysis Mode column of the well table in the Analysis screens In Protein Thermal Shift studies a value that indicates how tightly the fluorescence data within the ROA corresponds to the Boltzmann equation In Protein Thermal Shift studies the melting temperature C calculated by fitting data in the region of analysis to the Boltzmann equation In Protein Thermal Shift studies a set of values to define a component of the protein melt reaction For example the default conditions in the Protein Thermal Shift Software are Protein Ligand Buffer and Salt You can define the conditions and the condition values in the Setup gt Conditions screen of the software In Protein Thermal Shift studies the melting temperature C calculated for the region of analysis using the derivative of the melt curve An electronic record that contains all information abo
83. perature and the start or end of the Boltzmann fit curve The gap occurs if the defined ROA start or end temperature does not correspond exactly with a fluorescence datapoint Review the melt curves to visualize the fluorescence and derivative fluorescence data in the example ligand titration study Note For NPC wells the derivative melt curves are not displayed 1 Inthe Analysis gt Well Results screen select Ve Show in Plot then select to show these plot components e Unselected Wells e Legend 2 Select if Color By then select Ligand to color the melt curves according to the ligand condition value assigned for the well Note To set the color for each ligand to make the plots easier to distinguish go to the Setup gt Conditions screen 3 For each replicate group select all the wells in the replicate group then review the fluorescence levels in the melt curves e For the NPC wells do you observe a rise in fluorescence If so the wells or protein melt reactions may be contaminated with protein or the dye may interact with a buffer component e For the LOC wells do you observe a rise in fluorescence A rise in fluorescence in LOC wells but not in NPC wells may indicate protein contamination in the ligand or ligand dye interactions Protein Thermal Shift Studies User Guide 25 Getting Started with a Protein Thermal Shift Study Review the well results About the analysis mode Set the analysis mode 26 For samp
84. plicate results Review the Tm statistics 46 Using the Protein Thermal Shift Software review the Tm statistics for the replicate groups and look for positive hits in the Analysis gt Replicate Results screen This section provides guidance on how to review and interpret the replicate results Some common troubleshooting causes are provided For more detailed information on troubleshooting see page 83 In the Analysis gt Replicate Results screen review the Tm statistics to evaluate the variability among replicates The Tm statistics for each ROA are plotted along the x axis for each replicate group 1 In the Analysis gt Replicate Results screen select lt gt Plot by then select the type of Tm statistics to review e Tm Boltzmann e Tm Derivative Specify the condition hierarchy to group the replicate plots and change the order of conditions in the plot Note Changing the condition hierarchy does not affect the results it only changes how the replicate plots are grouped and the order in which the conditions are displayed in the Replicate Results Plot a Click Condition Hierarchy above the top right corner of the plot b In the dialog box select a condition then use the Up and Down arrows to change the hierarchy of conditions The condition at the top most level of the hierarchy is displayed on the far right side of the Replicate Results Plot and the replicate plots are grouped according to the top most co
85. rarchy does not affect the results it only changes how the replicate plots are grouped and the order in which the conditions are displayed in the Replicate Results Plot a Click Condition Hierarchy above the top right corner of the plot b In the dialog box select a condition then use the Up and Down arrows to change the hierarchy of conditions c For the example ligand titration study continue to move the conditions up and down until you obtain this hierarchy Condition Hierarchy wy Specify the hierarchy and change the order of conditions in the plot Analysis Group Buffer Protein Ligand The condition at the top most level of the hierarchy is displayed on the far right side of the Replicate Results Plot Ligand Protein Buffer Analysis Group 3 Scan the Replicate Results Plot to review the conditions that affect the Tm values relative to the reference replicate group Reference replicate plot red Sample replicate plot blue Sample replicate plot blue 3 4045 50 5 60 Temperature C 30 Protein Thermal Shift Studies User Guide Chapter 1 Getting Started with a Protein Thermal Shift Study Review the replicate results 4 Review the Tm statistics for each replicate group in the Replicate Results Plot a Zoom in Click then click drag an area on the plot one or more times am b Move the plot Click gt then click drag the plot until the replicate plot of interest is in
86. reate and set up an experiment file for the instrument run Setup Setting Run method e Reaction Volume Per Well 20 pL e Thermal profile Step 1 Temp 25 C Time 2 minutes Step 2 Temp 99 C Time 2 minutes e Ramp mode Continuous e Ramp rate ViiA 7 System Step 1 1 6 C s Step 2 0 05 C s StepOne and StepOnePlus Systems and 7500 and 7500 Fast Systems 1 e Optical Filters ViiA 7 System only Excitation Filter x4 580 10 Emission Filter m4 623 14 Defining and We recommend that you define and assign targets so you can review the melt curves assigning targets for the replicate groups in the Real Time PCR System Software before importing the experiment files into the Protein Thermal Shift study Define the following targets One target for each ligand or for each ligand titration e Define a target for the no protein control NPC e Define a target for the ligand only control LOC IMPORTANT Make sure that the well assignments correspond exactly with the arrangement of reactions in the reaction plate Setup errors may result in an incorrect grouping of replicates Example targets In the ViiA 7 System ligand screening example file targets were defined for each concentration of ligand the ligand only control LOC and no protein control NPC Target Name Reporter Quencher Color ProteinA 0 mM L ROX wv None v Example pl
87. rmation about how to use the software refer to the Protein Thermal Shift Software Help Setup guidelines The experiment files that you import into the study must contain analyzed melt curve data from a complete melt curve run e Set up the analysis group so that it contains experiment files from only one instrument Create and set up 1 In the Home screen of the Protein Thermal Shift Software click amp Create the study Study 2 Complete the Setup gt Properties screen e The Study Name cannot be more than 100 characters and cannot contain these characters V lt gt 1 The instrument selection must match the instrument type that you used to run the protein melt reactions and generate the experiment files 3 In the Setup gt Conditions screen define the conditions and condition values then list the analysis groups for your study 56 Protein Thermal Shift Studies User Guide Review the analysis settings 8 Chapter 3 Mutation Screening Studies 3 Set up the analysis In the Setup gt Experiment Files screen click P Import then select the experiment file eds for the instrument type that you selected for the study Repeat for each experiment file to import into the study Note The experiment file name and data are imported Plate setup information is not imported For each experiment file assign the conditions task and analysis group to the wells that contained protein melt reactions
88. rofits loss of good will loss of business or other financial loss or personal injury or property damage No agent employee or representative of Life Technologies has any authority to modify the terms of this Limited Warranty Statement or to bind Life Technologies to any affirmation representation or warranty concerning the product that is not contained in this Limited Warranty Statement and any such modification affirmation representation or warranty made by any agent employee or representative of Life Technologies will not be binding on Life Technologies unless in a writing signed by an executive officer of Life Technologies This warranty is limited to the buyer of the product from Life Technologies and is not transferable Some countries or jurisdictions limit the scope of or preclude limitations or exclusion of warranties of liability such as liability for gross negligence or willful misconduct or of remedies or damages as or to the extent set forth above In such countries and jurisdictions the limitation or exclusion of warranties liability remedies or damages set forth above shall apply to the fullest extent permitted by law and shall not apply to the extent prohibited by law Protein Thermal Shift Studies User Guide Ordering Information For more information on the Protein Thermal Shift reagents and Protein Thermal Shift Software go to www appliedbiosystems com Table 1 Products for Protein Thermal Sh
89. rt For systems that have built in computers or processing units installing unauthorized hardware or software may void the Warranty or Service Plan Limited product warranty Limited warranty Warranty period effective date Life Technologies warrants that for a period of ninety 90 days from the date the warranty period begins its Protein Thermal Shift Software v1 0 will perform substantially in accordance with the functions and features described in its accompanying documentation when properly installed on the instrument system for which it is designated and that for a period of ninety 90 days from the date the warranty period begins the tapes diskettes or other media bearing the software product will be free of defects in materials and workmanship under normal use If buyer believes that it has discovered a failure of the software to satisfy the foregoing warranty and if buyer notifies Life Technologies of such failure in writing during the ninety 90 day warranty period and if Life Technologies is able to reliably reproduce such failure then Life Technologies at its sole option will either i provide any software corrections or bug fixes of the identified failure if and when they become commercially available to buyer free of charge or ii notify buyer that Life Technologies will accept a return of the software from the buyer and upon such return and removal of the software from buyer s systems terminate the license t
90. s 1 2 Click Ba Analysis Settings in the toolbar On the Positive Hit tab specify the ATm Boltzmann and ATm Derivative values to indicate a positive hit e Select gt to identify buffer conditions that increase protein thermal stability or select lt to identify buffer conditions that decrease protein thermal stability Enter the number of degrees C of Tm shift relative to the reference to indicate a positive hit On the Flags tab specify settings for applying flags a Select the flags to use in the analysis b For the High Background High NPC Low Signal and Poor Fit flags specify the condition and threshold for applying the flag Apply the analysis settings and analyze e Click Apply to apply the analysis settings and reanalyze while keeping the Analysis Settings dialog box open or e Click OK to apply the analysis settings reanalyze and close the Analysis Settings Protein Thermal Shift Studies User Guide 41 2 Buffer Screening Studies Review the well results Review the well results Review the melt curves 42 Using the Protein Thermal Shift Software review the melt curves and well table and optimize the analysis in the Analysis gt Well Results screen The Well Results screen displays fluorescence and derivative melt curve plots calculated Tm values individual well results and flags As necessary edit the analysis settings edit the baseline edit the region of analysis edit the analysis mode
91. s MicroAmp Optical Reaction Plate appropriate for your real time PCR instrument MicroAmp Optical Adhesive Film appropriate for your reaction plate Prepare the protein We recommend that you prepare four replicates of each reaction melt reactions 1 3 4 54 Prepare a fresh dilution of Protein Thermal Shift Dye 1000X to 8X 2 Place the appropriate reaction plate or tubes on ice then prepare the protein melt reactions e Make sure that the arrangement of reactions in the reaction plate corresponds exactly with the well assignments in the experiment file IMPORTANT Setup errors may result in an incorrect grouping of the data and incorrect Tm statistics e Add reaction components to the plate in the order listed Component Volume Protein Thermal Shift Buffer 5 0 ul Water protein 12 5 uL Diluted Protein Thermal Shift Dye 8x 2 5 uL Total volume for each control reaction 20 0 pl Pipet each reaction up and down 10 times to mix well Seal the plate with MicroAmp Optical Adhesive Film spin it at 1000 rpm for 1 minute then place it on ice Protein Thermal Shift Studies User Guide Chapter 3 Mutation Screening Studies Run the protein melt reactions Run the protein melt reactions Load and run the reactions Review the melt curves Load and run the protein melt reactions on a supported Applied Biosystems Real Time PCR System then analyze and save the experiment fil
92. s are dissimilar pipetting errors may have occurred during reaction setup or condition assignments may be incorrect 4 Ifthe derivative melt curves for the replicate group show multiple melt phases set the analysis mode to Auto Multiple Tm then review the derivative melt curves a In the Well Table or in the melt curve plot select the wells with multiple melt phases b Click P Auto Analysis Options then select Auto Multiple Tm c Click gt Analyze to reanalyze using the Auto Multiple Tm analysis mode d Review the number of melt phases in the derivative melt curves e Doall replicate groups have the same number of melt phases e For each replicate group are there outliers with a different number of melt phases than the other samples in the replicate group You may consider omitting outliers from analysis Review the regions Review the ROAs detected by the software If necessary edit the ROAs of analysis ROA Note If no melt phases are detected no ROAs are defined Negative controls should have no melt phases and no ROAs 1 Inthe Analysis gt Well Results screen confirm that each ROA meets the following criteria e For melt curves with one melt phase the curve within the ROA resembles a sigmoidal profile e Atthe start temperature the signal is relatively flat e Atthe end temperature the signal has already reached its maximum 2 For each replicate group edit the ROAs so that all of the wells in the replic
93. six ROAs for each melt curve If you edit or delete an ROA detected by the software the analysis mode is changed to Manual In the example ligand titration study the melt curves should show one melt phase If you see more than one peak the wells or protein melt reactions may be contaminated with protein In your own studies you may observe multiple melt phases If the derivative melt curves for the replicate group show multiple melt phases set the analysis mode to Auto Multiple Tm then review the derivative melt curves 1 In the Well Table or in the melt curve plot select the wells with multiple melt phases gt Click M Auto Analysis Options then select Auto Multiple Tm Click gt Analyze to reanalyze using the Auto Multiple Tm analysis mode Review the number of melt phases peaks in the derivative melt curves Do all replicate groups have the same number of melt phases For each replicate group are there outliers with a different number of melt phases than the other samples in the replicate group You may consider omitting outliers from analysis Protein Thermal Shift Studies User Guide Chapter 1 Getting Started with a Protein Thermal Shift Study Review the well results Review the regions Review the ROAs detected by the software If necessary edit the ROAs of analys is ROA Note If no melt phases are detected no ROAs are defined Negative controls should have no melt phases and no ROAs
94. t Buffer 5 0 uL Water protein buffer and or buffer 12 5 uL components Diluted Protein Thermal Shift Dye 8x 2 5 uL Total volume for each control reaction 20 0 pl 3 Pipet each reaction up and down 10 times to mix well 4 Seal the plate with MicroAmp Optical Adhesive Film spin it at 1000 rpm for 1 minute then place it on ice 38 Protein Thermal Shift Studies User Guide Chapter 2 Buffer Screening Studies Run the protein melt reactions Run the protein melt reactions Load and run the reactions Review the melt curves Load and run the protein melt reactions on a supported Applied Biosystems Real Time PCR System then analyze and save the experiment file before importing the experiment file into the Protein Thermal Shift Software IMPORTANT Keep the protein melt reactions on ice until you load the instrument 1 Ifnecessary transfer the experiment file that you created for the run to the computer that is connected to the instrument 2 In the Home screen of the Real Time PCR System Software click Open then select the experiment file you created for the run 3 Load the reaction plate into the instrument 4 In the Real Time PCR System Software click Run in the navigation pane then click START RUN Using the Real Time PCR System Software open the experiment file from the completed instrument run analyze and save the experiment file then review the melt curves Note You must analyze
95. t file for the instrument run Create and set up an experiment file for the instrument run Create and set up an experiment file for the ViiA 7 System 12 Define and assign targets so you can review the melt curves for the replicate groups in the ViiA 7 Software 1 Select Start gt All Programs gt Applied Biosystems gt ViiA 7 Software gt ViiA 7 Software v1 1 Note If this is your first time starting the software read and accept the license agreement 2 In the Home screen of the ViiA 7 Software click Experiment Setup 3 Complete the Experiment Properties screen Field Entry Experiment Name Enter a unique Experiment Name using up to 100 letters and or numbers Note If you plan to start the run using the instrument touchscreen do not enter more than 32 characters for the Experiment Name and do not include spaces in the name Block type Select the type of block that you are using 384 Well Block or 96 Well Block 0 2mL Experiment type Melt Curve Reagent type Other Ramp speed Fast 4 In the Define screen define the targets conditions and dyes a b In the Targets pane enter the target name for each condition select ROX for the reporter and select None for the Quencher For the example ligand titration study Target Name Reporter Quencher Color ProteinA 0 mM L ProteinA 0 1 mM L ProteinA 1 mM L Loc NPC
96. t form oligomers you can perform a buffer additive screening study to identify conditions in which the protein unfolding follows a two state model Protein Thermal Shift Studies User Guide Getting Started with a Protein Thermal Shift Study General guidelines Experimental conditions Plate layout Replicates Controls 10 We recommend that you begin with the experimental conditions that we provide If you do not observe clear well resolved melt phases in the melt curves and a reasonable rise in fluorescence relative to the NPC wells you can optimize the experimental conditions according to your protein e Protein dye ratio If you observe high initial fluorescence or small transitional increase in your signal then we recommend that you perform a titration study with the protein and Protein Thermal Shift Dye to identify the optimal protein dye ratio Ramp speed and ramp rate Start with the Fast ramp speed and the recommended ramp rate then decrease the ramp speed and or ramp rate as needed to obtain well resolved melt phases In Protein Thermal Shift studies the ramp speed affects protein unfolding and the resulting melt curves With some proteins we have observed low resolution of the melt phases in the melt curves with the Fast ramp speed and high ramp rates For these proteins we observed improved resolution of the melt phases when we used the Standard ramp speed and decreased ramp rates Thermal prof
97. t side of the Replicate Results Plot Scan the Replicate Results Plot to review the conditions that affect the Tm values relative to the reference replicate group Reference replicate plot red Sample replicate plot blue Sample replicate plot blue Protein Thermal Shift Studies User Guide Chapter 4 Ligand Screening Studies Review the replicate results 4 Review the Tm statistics for each replicate group in the Replicate Results Plot a Zoom in Click then click drag an area on the plot one or more times am b Move the plot Click gt then click drag the plot until the replicate plot of interest is in view c Place the cursor within the diamond then wait to view a tooltip with the Tm statistics for the replicate group Tm Statistics Mean 43 75 Lower 95 43 67 Upper 95 43 83 Median 43 77 Std Error 0 04 Min 42 96 Max 43 90 d To examine outliers place the cursor over a datapoint then wait to view a tooltip with the well information experiment file name and the Tm selected for the plot Well B21 Experiment Ligand_Screening_Example_ViiA7 eds Tm Boltzmann 43 35 Note Click I to restore the default zoom 5 In the Replicate Groups table review the Tm statistics for each replicate group e Std Error standard error of the mean for the Tm value Is the value low If the value is high review the data for each replicate e Min and Max minimu
98. t the start temperature the signal is relatively flat e Atthe end temperature the signal has already reached its maximum 2 For each replicate group edit the ROAs so that all of the wells in the replicate group have the same ROAs a Select the replicates click Define ROA in the toolbar above the melt curve plots then click drag an area in one of the plots to define a melt phase and replace the ROA Repeat for each melt phase you identify b If necessary adjust the start and end temperatures for each ROA To move the ROA Starting from within the ROA click drag the ROA e To adjust the start and end temperatures individually Click drag the ROA start or the end line Protein Thermal Shift Studies User Guide 43 2 Buffer Screening Studies Review the well results IMPORTANT Make sure that the fluorescence at the start temperature is lower than the fluorescence at the end temperature 3 Click gt Analyze to reanalyze using the edited ROAs Note After you edit ROAs the analysis mode changes from Auto to Manual Review the flags Review the flags and Tm values then consider editing the analysis settings and or and Tm values omitting wells before you review the replicate results 1 Inthe Analysis gt Well Results screen select LE Show in Plot then select to show these plot components e Boltzmann Tm e Derivative Tm e Unselected Wells e Legend 2 Review flagged wells in the Well Table a Click the
99. te file csv The example files are located in lt drive gt Program Files Applied Biosystems Protein Thermal Shift Software examples where lt drive gt is where you installed the software 1 Go to the Setup gt Experiment Files screen click Import then select the ligand screening example file eds for the instrument type that you selected for the study e Ligand_Screening_Example_ViiA7 eds e Ligand_Screening_Example_StepOnePlus eds e Ligand_Screening_Example_7500 eds Note The experiment file name and data are imported Plate setup information is not imported Click a Load Plate Template then select the ligand screening plate template file csv for the instrument type that you selected for the study e Ligand_Screening_Example_Setup_ViiA7 csv e Ligand_Screening_Example_Setup_StepOnePlus csv e Ligand_Screening_Example_Setup_7500 csv The conditions task and analysis group are assigned to the wells that contained protein melt reactions Make sure that the condition assignments correspond exactly with the contents of the reaction plate IMPORTANT Setup errors may result in an incorrect grouping of the data and incorrect Tm statistics Select the replicate group to use as the reference a Select the wells that contain 0 1 mM ligand Instrument type Select wells ViiA 7 System Click the B row header to select row B wells B1 B24 StepOnePlus System Click drag the 4 column header
100. te group in the Replicate Results Plot a Zoom in Click then click drag an area on the plot one or more times am b Move the plot Click gt then click drag the plot until the replicate plot of interest is in view c Place the cursor within the diamond then wait to view a tooltip with the Tm statistics for the replicate group Tm Statistics Mean 43 75 Lower 95 43 67 Upper 95 43 83 Median 43 77 Std Error 0 04 Min 42 96 Max 43 90 d To examine outliers place the cursor over a datapoint then wait to view a tooltip with the well information experiment file name and the Tm selected for the plot Well B21 Experiment Ligand_Screening_Example_ViiA7 eds Tm Boltzmann 43 35 Note Click I to restore the default zoom 5 In the Replicate Groups table review the Tm statistics for each replicate group e Std Error standard error of the mean for the Tm value Is the value low If the value is high review the data for each replicate e Min and Max minimum and maximum Tm values for the replicate group Is the range of Tm values for the replicate group within 1 degree If the range of Tm values exceeds 1 degree review the data for each replicate 6 Omit outliers as necessary then click p Analyze Protein Thermal Shift Studies User Guide 63 3 Mutation Screening Studies Review the replicate results Review the positive In the Analysis gt Replicate Results screen
101. tein page 86 for conditions that increase Thermal Shift studies 98 C or thermal stability of the protein higher e You may consider performing Protein Thermal Shift studies to screen for buffers ligands or mutations that decrease thermal stability of the protein Protein solution contains high levels High initial background signal page 85 Perform protein dye titration of detergent gt 0 02 studies to optimize the protein High fluorescence in NPC wells i A concentration and protein dye ratio e Repurify the protein using an ammonium sulfate precipitation method Resolubilize the purified protein using HEPES buffer or a buffer with neutral pH then add glycerol and DTT Buffer component interacts with the High initial background signal page 85 Perform protein dye titration aye High fluorescence in NPC wells studies to optimize ne protein concentration and protein dye ratio Bien Noe LE Wels e Perform a buffer screening Protein Thermal Shift study to identify alternative buffer conditions Ligand interacts with the dye High initial background signal page 85 Use an alternate method to screen for conditions that affect thermal stability High fluorescence in LOC wells of the protein Protein aggregation or the protein is High initial background signal page 85 Repeat the study with a fresh partially unfolded Flat signal or decrease in signal protein sample page 8
102. that is necessary for proper instrument operation or accurate chemistry kit use CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury Protein Thermal Shift Studies User Guide 5 About This Guide User attention words 6 Protein Thermal Shift Studies User Guide Getting Started with a Protein Thermal Shift Study Perform a Protein Thermal Shift study to screen for buffers mutations or ligands that affect the thermal stability of the protein of interest With Protein Thermal Shift studies you can determine optimal protein storage conditions perform functional characterization or improve the success rates of protein purification and crystallization This chapter provides instructions for using the Protein Thermal Shift Starter Kit to perform an example ligand titration study In the example ligand titration study you titrate a ligand to determine the concentration that increases the thermal stability of the protein Perform an example ligand titration study Create and set up an experiment file for the instrument run e ViiA 7 System page 12 e StepOne or StepOne
103. the replicate group 76 Protein Thermal Shift Studies User Guide Review the well results Chapter 4 Ligand Screening Studies Note When you define the ROA manually you may observe a gap between the ROA start or end temperature and the start or end of the Boltzmann fit curve The gap occurs if the defined ROA start or end temperature does not correspond exactly with a fluorescence datapoint Example well For the ViiA 7 System example buffer screening study observe the following results For the ViiA 7 System example ligand screening study observe the following Flourescence levels are flat in the NPC and LOC wells For the sample and reference replicate groups the fluorescence levels as displayed in the fluorescence melt curve are not significantly different so you do not need to edit the baseline The sample and reference wells contain one peak in the derivative melt curve Within each replicate group The fluorescence melt curves and the Boltzmann Tm values are similar The derivative melt curves and the derivative Tm values are similar and there are no outliers For each well the region of analysis meets the recommended criteria Protein Thermal Shift Studies User Guide 77 4 Ligand Screening Studies Review the replicate results Review the replicate results Review the Tm statistics 78 Using the Protein Thermal Shift Software review the Tm statistics for the replicate groups and look f
104. the run If the protein is thermally stable at ambient temperatures you may consider preparing the reactions in advance and leave the reaction plate at ambient temperature protected from light However the fluorescence levels will decrease over time and the Tm values will vary depending on the protein and its thermal stability If you want to prepare the reaction plates in advance we recommend that you first determine the benchtop stability of your protein melt reactions Required materials Required materials for protein melt reactions e Protein Thermal Shift Dye 1000X e Protein Thermal Shift Buffer e Water e Protein e Buffers and or buffer components MicroAmp Optical Reaction Plate appropriate for your real time PCR instrument e MicroAmp Optical Adhesive Film appropriate for your reaction plate Prepare the protein We recommend that you prepare four replicates of each reaction melt reactions 1 Prepare a fresh dilution of Protein Thermal Shift Dye 1000X to 8X 2 Place the appropriate reaction plate or tubes on ice then prepare the protein melt reactions e Make sure that the arrangement of reactions in the reaction plate corresponds exactly with the well assignments in the experiment file IMPORTANT Setup errors may result in an incorrect grouping of the data and incorrect Tm statistics e Add reaction components to the plate in the order listed Component Volume Protein Thermal Shif
105. the well Note To set the color for each protein variant to make the plots easier to distinguish go to the Setup gt Conditions screen Protein Thermal Shift Studies User Guide Review the well results Chapter 3 Mutation Screening Studies 3 For each replicate group select all the wells in the replicate group then review the fluorescence levels in the melt curves e For the NPC wells do you observe a rise in fluorescence If so the wells or protein melt reactions may be contaminated with protein or the dye may interact with a buffer component e For sample or reference wells do you observe flat melt curves If so condition assignments may be incorrect or a component is missing from the protein melt reactions e Within each replicate group are the fluorescence melt curves similar to each other Within each replicate group are the derivative melt curves similar to each other If the melt curves for the replicates are dissimilar pipetting errors may have occurred during reaction setup or condition assignments may be incorrect 4 Ifthe derivative melt curves for the replicate group show multiple melt phases set the analysis mode to Auto Multiple Tm then review the derivative melt curves a In the Well Table or in the melt curve plot select the wells with multiple melt phases b Click Auto Analysis Options then select Auto Multiple Tm c Click gt Analyz
106. the well results page 58 l Review the replicate results page 62 Set up the analysis page 56 Example experiment files and plate template files are located in the examples folder lt drive gt Program Files Applied Biosystems Protein Thermal Shift Software examples where lt drive gt is where you installed the software To view the data for the buffer screening example used in this chapter use the Mutation_Screening_Example_ViiA7 eds and Mutation_Screening_Example_Setup_ViiA7 csv files in the ViiA7 Example Files folder Protein Thermal Shift Studies User Guide 51 3 Mutation Screening Studies General guidelines General guidelines For general guidelines that apply to all Protein Thermal Shift studies see page 9 Experimental Before you perform a mutation screening study we recommend that you first perform design a buffer screening study to identify a buffer in which the wild type protein is thermally stable Replicates and For mutation screening studies we recommend that you prepare controls e Atleast 4 replicates of each reaction e At least 4 replicates of no protein controls NPCs Create and set up an experiment file for the instrument run This section contains general settings for creating and setting up an experiment file For detailed instructions see Instrument Page ViiA 7 Real Time PCR System page 12 StepOne and StepOnePlus Real Time PCR Systems pa
107. tive melt curves ROA start ROA end The fluorescence melt curves show noise reduced fluorescence data from the protein melt curve run The fluorescence melt curves and the Boltzmann method are used to determine the Boltzmann Tm The derivative melt curves are calculated using a first derivative of the fluorescence data at each temperature The derivative melt curve is used to identify melt phases or regions of analysis ROAs from which the derivative Tm is calculated In Protein Thermal Shift Software up to six ROAs and derivative Tm values can be automatically detected or manually defined for each melt curve Because the Boltzmann Tm and derivative Tm values are calculated independently of each other the values may be dissimilar Protein Thermal Shift Studies User Guide About the Boltzmann fit curve Review the melt curves Review the well results Chapter 1 Getting Started with a Protein Thermal Shift Study The Boltzmann fit curve is calculated for the region of analysis ROA according to the Boltzmann equation and is plotted in the fluorescence melt curve plot as a dark green curve Fluorescence melt curve Boltzmann fit curve dark green 325 350 375 400 425 450 475 8500 525 The Boltzmann fit value corresponds to how similar the fluorescence melt curve and the Boltzmann fit curve are Note When you define the ROA manually you may observe a gap between the ROA start or end tem
108. to select columns 4 or 7500 Fast System 6 wells A4 A6 B4 B6 C4 C6 D4 D6 E4 E6 F4 F6 G4 G6 H4 H6 b Click RZ Assign then select Reference as the Task aes 5 Click fy Save in the toolbar to save and analyze the study With the analysis settings you can adjust how positive hits are determined and specify settings for applying flags If you generate a study template from the study file the analysis settings for the study are saved in the study template Although the flags and the positive hits can be useful ways to quickly scan the results we recommend that you review the values in the tables review the plots and review the replicates carefully Protein Thermal Shift Studies User Guide Chapter 1 Getting Started with a Protein Thermal Shift Study 1 Set up the analysis Review the analysis Review and edit the analysis settings to optimize the analysis for your study settings 1 2 Click H Analysis Settings in the toolbar On the Positive Hit tab specify the ATm Boltzmann and ATm Derivative values to indicate a positive hit e Select gt to identify conditions that increase protein thermal stability or select lt to identify conditions that decrease protein thermal stability Enter the number of degrees C of Tm shift relative to the reference to indicate a positive hit On the Flags tab specify settings for applying flags a Select the flags to use in the analysis b For the High Background
109. ubleshooting 5 Examples of symptoms Examples of symptoms High initial background signal In this example protein samples with high detergent levels produce a high initial background signal in the melt curves Figure 1 Melt curves in ViiA 7 Software High initial background signal in protein samples with high detergent levels 000 0 20000 Normal melt curves in protein samples with acceptable detergent levels Normalzed Reporter Rn No rise in fluorescence levels in NPC wells some SIGHTS AST eng Sehnert renin High initial background signal in protein samples with high detergent levels Normal melt curves in protein samples with acceptable detergent levels ia N0o rise in fluorescence 25 30 38 40 p 30 55 CJ o5 70 75 80 85 80 85 levels in NPC wells Protein Thermal Shift Studies User Guide 85 5 Troubleshooting Examples of symptoms Flat signal or In this example flat signals in the melt curves are observed because the passive decrease in signal reference is not set to None Passive Melt curves in StepOne Software Melt curves in Protein Thermal Shift Software reference TAMRA sooo v0000 70000 20 5000 0 t j 00 20 f o 30000 Fluorescence Normaizea Reporter kn 10000 u kuorescanceya a 2 gt o o s a s oF 7 7 o amp oo amp 100 Temperature C
110. using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document Chemical safety WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions e Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood e Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS e Handle chemical wastes in a fume hood e Charact
111. ut a particular plate including metadata name barcode comments plate setup well contents assay definitions run method thermal profile run results analysis settings analysis results and other plate specific data The analysis settings and analysis results in the experiment files are limited to the protein melt data from the real time PCR instrument The experiment files do not contain any settings or results from the protein thermal shift analysis Experiment files have the suffixes eds experiment document single edt template and edm multiple Protein Thermal Shift Studies User Guide 97 Glossary export LOC melt curve plot melt phase melting temperature Tm NPC omit well outlier plate layout positive hit 98 A software feature that allows you to save raw data and analysis results to txt or csv files You can select which data to export the file format and the file name and location Ligand Only Control Protein melt reactions that contain only ligand buffer water and dye A plot of fluorescence data collected during the melt curve stage Melt curve plots are displayed in the real time PCR software and the Protein Thermal Shift Software The melt curves in the real time PCR software may not exactly match the melt curves in the Protein Thermal Shift Software When the experiment files are imported into the Protein Thermal Shift Software the Protein Thermal Shift Soft
112. view c Place the cursor within the diamond then wait to view a tooltip with the Tm statistics for the replicate group Tm Statistics Mean 43 75 Lower 95 43 67 Max 43 90 d To examine outliers place the cursor over a datapoint then wait to view a tooltip with the well information experiment file name and the Tm selected for the plot Well B21 Experiment Ligand_Screening_Example_Viid7 eds Tm Boltzmann 43 35 Note Click I to restore the default zoom 5 In the Replicate Groups table review the Boltzmann Tm statistics for each replicate group e Tm B Std Error standard error of the mean for the Boltzmann Tm Is the value low If the value is high review the data for each replicate e Tm B Min and Tm B Max minimum and maximum Boltzmann Tm values for the replicate group Is the range of Boltzmann Tm values for the replicate group within 1 degree If the range of Tm values exceeds 1 degree review the data for each replicate 6 Omit outliers as necessary then click p Analyze Protein Thermal Shift Studies User Guide 31 Getting Started with a Protein Thermal Shift Study Review the replicate results Review the positive In the Analysis gt Replicate Results screen review the positive hits to identify the hits conditions that produce the maximum effect on thermal stability Replicate groups with positive hits have ATm values that exceed the threshold set in th
113. ware reduces the noise in the fluorescence data TM In the Protein Thermal Shift Software the shape of the melt curve can indicate the protein thermal stability or multiple states of protein ligand binding You can view the melt curve as determined by fitting the fluorescence data to the Boltzmann equation in single Tm mode or as a differential melt curve in single Tm mode or in multiple Tm mode In Protein Thermal Shift studies a region within the melt profile that shows a nearly continuous increase in fluorescence followed by a noticeable decrease in stabilization of fluorescence levels A melt profile may show one or more melt phases The temperature C at which 50 of the protein is folded and 50 of the protein is melted The Tm values are displayed in the melt curve plots No Protein Control Protein melt reactions that contain only buffer water and dye An action that you perform in the Protein Thermal Shift Software before reanalysis to omit one or more wells from calculations Omitted wells are analyzed and can be displayed in the Replicate Results plot The results are hidden from the results tables and are not used to calculate Tm statistics for the associated replicate group You can add wells back to the analysis no information is permanently discarded A measurement that deviates significantly from the measurement of the other replicates in the replicate group An illustration of the grid of wells and assign
114. well in the Well Table then compare the fluorescence melt curve to the Boltzmann fit curve dark green thick curve and review the value in the B Fit Boltzmann Fit column of the Well Table e How well does the fluorescence melt curve correspond to the Boltzmann fit curve e Is the B Fit value close to 1 e Is the B Fit value similar among wells in the replicate group Note When you define the ROA manually you may observe a gap between the ROA start or end temperature and the start or end of the Boltzmann fit curve The gap occurs if the defined ROA start or end temperature does not correspond exactly with a fluorescence datapoint Example well For the ViiA 7 System example buffer screening study observe the following results e Fluorescence levels are flat in the NPC wells For the sample and reference replicate groups the fluorescence levels as displayed in the fluorescence melt curve are not significantly different so you do not need to edit the baseline The sample and reference wells contain one peak in the derivative melt curve Within each replicate group The fluorescence melt curves and the Boltzmann Tm values are similar The derivative melt curves and the derivative Tm values are similar and there are no outliers For each well the region of analysis meets the recommended criteria Protein Thermal Shift Studies User Guide 45 2 Buffer Screening Studies Review the replicate results Review the re
115. y and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensingfalifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Microsoft and Windows are registered trademarks of Microsoft Corporation Intel and Pentium are registered trademarks of Intel Corporation 2011 Life Technologies Corporation All rights reserved Part Number 4461808 Rev A May 2011 Contents About This Guides cicieiiince ceed daw hade anne oe RPM Re ee ewe eek 5 PUrposeLssnastr testet audaner ute poet aah PRRs Sted wheal Beto Roamer arealene 5 User ttentionwords aus c 0502 Atco sien Bea kari Lanta ea nan cated wie tania sand Arla 5 M CHAPTER 1 Getting Started with a Protein Thermal Shift Study 7 Product information setrini aro saa dale ana da eae nest eae ea ote pean da lek 8 General guidelines 0 0 0 0 cece eect tte n ence eee eee eens 9 Create and set up an experiment file for the instrument run 0202000 0 cece eee 12 Prepare the protein melt reactions unnn cece eee eee eee e eee aes 18 Run the protein melt reactions

Protein Thermal Shift™ Studies User Guide

Contents

Download Pdf Manuals

Related Search

Related Contents

Protein Thermal Shift&trade; Studies User Guide

Contents

Download Pdf Manuals

Related Search

Related Contents

Protein Thermal Shift™ Studies User Guide