Optimizer User Guide
Contents
1. Materials needed Q 400 mL of 1 X Separation Buffer with DMSO for example 100 mM HEPES pH 7 3 0 015 Brij 35 5 0 DMSO 0 1 Coating Reagent 3 1mM EDTA 2mL of 10uM CR 8 Caliper 760278 Note it is important to accurately match the composition of the separation buffer and the buffer in the chip and the buffer used for the samples on the plate Q 1to 2 uL of substrate stock solution gt 500 uM Q Electrically neutral dye For example Rhodamine B Sigma 25 242 5 or Bodipy Fluorescein Arginine included in Caliper LabChip Test Kit PN 760403 Q Product solution prepared above Section 1 4 Q Caliper Mobility Shift assay chip EZ Reader 4 or 12 sipper at room temperature O O 0 OO OO Procedure 1 Prepare approximately 1 uM solutions of the substrate product and neutral dye Also prepare a mixture of substrate and product at about 1 uM each If using an EZ Reader 4 sipper chip make 300 uL of each solution If using an EZ Reader 12 sipper chip make 1 2 mL of each solution Caliper Life Sciences Inc 4 Optimizer for Mobility Shift Assays Step By Step Guide to Using the Optimizer Spreadsheet 2 Plate Preparation Prepare a Greiner 384 well microtiter plate as illustrated in Figure 2 for use with 4 sipper chips or Figure 3 for use with 12 sipper chips where o N 70ulof neutral dye 1 uM Bodipy Fluorescein Arginine or 150 uM Rhodamine B o Sub 70 ul of 1 uM substrate peptide in separation buffer o Prod 72. 3 2 Example using only 4 sippers of a 12 Sipper Chip ccccceeeceeeeeceeeeeeeeeeneeeeeeeeeseeetneeeeeeeeteees 12 3 3 Review the Viscosity Mobility Measurement Results 0 0 02 eeeeeeeeeeeneeeeeeeeneeeeeeeaeeeeeenaeeeeeenaeeeeeeaas 13 Appendix A Mobility Shift Assay Predictions Worksheet c ccceceeeeeeeeeeeeeeeeeaeeeeeeaeeeeeeaeeeeeenaeeeeseaas 15 A 1 Selecing the Resolor iA bas cute adic duane duddevaducsuat nad EA todas sues tacts 15 A 1 1 How is Resolution Defined iesin iiuair ananin ad aaa aaa aa iaa aiaa 16 A 1 2 Practical Hints on Selecting the Resolution ect ee eene eter entree ee eaeeeeetaeeeeetaeeeeesnaeeeeee 17 A2 Troubleshooting Resolution Errors ccccccssceceesseeeceeseeeeceeseececeeneneeceeseneeceeneaeeseeseneeeeeneeeeseensaee 17 A 3 Interpreting the Input Data Table ccccccceceeeeeenee cece eeeeeeeenaeceeeeeeesecaaaeceeeeeseseccanaeeeeeeeeeteeaees 19 A 4 Interpreting the Output Data Table cccccccceeeeenecceeeeeeeeeceeaeceeeeeeesecacaeceeeeesesecsanaeeeeeeeeeneeaees 20 Appendix B Mobility Shift Calculator Worksheet ceceececeeeeeeeceneaeeeeeeeeeseceaeaeeeeeeesesensneeeeeeeneess 21 B 1 Definition f Output ValUeS vai sisccissccncdesseadcaivscnadcasvandanusantdacaasedd sade snchiceaveadsadevatdddenvandstdenanadceavaadstes 22 Copyright 2004 2012 Caliper Life Sciences Inc For Research Use Only Not for use in diagnostic procedures Caliper the
3. Accept button is clicked so you can see the results but there is nothing further to do on that sheet except view the results Run Auto Mode Analysis Macro Manual Mode C Projects EZ Reader 1 5 1400 1400 30 106 0 2 1 6 32 5 EZReader 4 Sipper Electro Mobilities cm2 V s Sub sere nEp Sub pEp Prod pp Prod a nEo 1st v nEo 2nd viscosity reson R st V 2nd V tst V 2nd V v Sipper to Detector Time s Factor 1 83 90 84 10 84 20 83 80 84 20 73 30 83 70 83 40 107 80 2 72E 06 2 09E 06 1 02E 04 1 00E 04 6 87E 07 1 38E 06 1 38 1 00 2 84 90 85 10 85 10 84 70 85 10 73 90 84 60 84 30 109 70 268E 06 2 06E 06 1 03E 04 1 01E 04 1 35E 06 2 02E 06 1 39 1 00 3 84 10 84 40 84 30 84 10 84 50 73 50 84 00 83 70 108 60 270E 06 207E 06 1 02E 04 1 01E 04 9 36E 11 6 83E 07 1 38 1 00 4 83 90 84 20 84 00 83 80 84 30 73 20 83 80 83 40 108 10 3 40E 06 2 79E 06 1 03E 04 1 00E 04 6 87E 07 6 87E 07 1 38 1 00 5 6 7 8 9 10 11 12 Mean 84 20 84 45 84 40 84 10 84 52 73 48 84 03 83 70 108 55 2 87E 06 2 25E 06 1 02E 04 1 01E 04 6 80E 07 1 19E 06 1 38 1 00 Results Output Estimated Substrate Product EO Electrical Substrate Product Mean Observed Observed Observed Observed Observed Observed Viscosity Mobility Mobility Mobility Resistance Retention Retention Current Currenti Current2 Current3 Current4 Current5 Current6 cP _ cm V s _ cm V s cm V s MO Factor Factor pA vA pA pA pA pA pA 1 38
4. Figure 13 Look for things like incomplete product formation sticky substrate product or viscosity outside of the expected range 3 Be sure that the buffer you used for the viscosity mobility measurement experiment is actually the same buffer that you are running the assay in If you change any of the ingredients in the buffer you must repeat the viscosity mobility measurement experiment using the new buffer 4 Control the Temperature Viscosity and mobility are temperature dependent Be sure that you perform the viscosity mobility experiment at the same temperature as you run the assay Be sure to allow the chip and buffer to reach the ambient temperature before performing the viscosity mobility measurement 5 Double check the Chip Type Be sure that you perform the viscosity mobility experiment using the same type of chip as you will use to run your assay For example if you want to run your assay using an EZ Reader 12 sipper chip you must use an EZ Reader 12 sipper chip to perform the viscosity mobility measurement experiment 6 Just repeat the viscosity mobility measurement experiment 7 f after considering the things listed above you still observe poor separation of peaks in the Separation Check file contact Caliper s Customer Service Group by calling 1 877 522 2447 for advice Be prepared to email us a copy of the spreadsheet you are using and the data file produced by your viscosity mobility measurement experiment Caliper
5. Results Auto Mode Filename Professor Professor D drive Hayley AppNote 2003 08 14 VISCMOB_CK2_2003 08 14_14 14 31 RDA Accept Review the charts and edit the table if needed then click Accept 1 135 3563 234 5563 334 0563 435 0563 578 6563 678 1563 767 0563 614 6563 914 0563 2 138 7563 237 9563 337 4563 438 5563 584 6563 684 1563 766 0563 817 4563 916 8563 3 134 7563 233 9563 333 3563 434 3563 576 1563 675 6563 767 2563 814 3563 913 7563 4 135 1563 234 3563 333 6563 434 8563 577 6563 677 1563 767 2563 814 6563 913 9563 x Type the times for the correct peak in this column then Click Accept Sipper 1 Time sec 1 6562 oe oe roren Figure 9 Example of a Viscosity Mobility measurement file that cannot be analyzed automatically by the spreadsheet due to the extraneous peak labeled near 800 sec The user needs to enter the arrival time of the correct Dye peak into the table above the plot and then click Accept The most common source of contamination that leads to extra interfering peaks in the viscosity mobility measurement data is contamination of the product by the substrate itself in other words incomplete product conversion see Figure 10 If the contamination of the product by the substrate is small enough that the peak finder ignores the substrate peak then the contamination will not interfere with the measurement and it can be safely ignored However if the product conversion
6. Several examples of Mobility Shift assay data to illustrate different resolutions of substrate and product peaks Appendix A Mobility Shift Assay Predictions Worksheet A 1 1 How is Resolution Defined B AT 2 0 0 Where AT is the distance in time between the two peaks and o is the standard deviation of the peak with the assumption that it is a Gaussian distribution These terms are illustrated in Figure 16 It is often difficult to estimate o1 and o2 from experimental data especially when the two peaks are partially overlapping such that the full width cannot be seen An alternative definition that uses parameters that are more easily observed is 2AT R 1 7 FWHM FWHM Where again AT is the distance in time between the two peaks and FWHM is the Full Width at Half Maximum value for the peak as illustrated in Figure 16 ob f f fi fi f f 1 f f 105 110 115 120 125 130 135 140 145 150 Figure 16 Illustration of terms for the definition of resolution used in Mobility Shift Assays Caliper Life Sciences Inc 16 Optimizer for Mobility Shift Assays Appendix A Mobility Shift Assay Predictions Worksheet A 1 2 Practical Hints on Selecting the Resolution When selecting the resolution for your particular assay there are several issues to consider i the minimum resolution required by the data analysis software is R 1 2 ii there will be some variability in the resolution over time iii the reso
7. The spreadsheet makes a recommendation as to which job file will make the best measurement of your peptide after you type in the sequence of your peptide on the A Priori Estimates worksheet see Figure 1 The experiment consists of a series of sample sips at varying pressure and voltage conditions You should see 9 peaks in each data trace The measurement job file takes about 18 minutes to run through to completion Caliper Life Sciences Inc 5 Optimizer for Mobility Shift Assays Step By Step Guide to Using the Optimizer Spreadsheet 2 6 Analyze the Data 1 Working in the Optimizer spreadsheet open the Experimental Estimates worksheet see Figure 4 2 Click the Run Analysis Macro button see Figure 4 and then enter the name of the data rda file that was created in Step 1 5 11 The analysis macro will fill in all of the input cells on this worksheet so you do not need to enter any data other than the file name 3 Review the data plot for each sipper in the Peak Finder Results worksheet shown in Figure 5 Check to be sure that the peak finder found all of the peaks correctly If you do not see a yellow marker on each of the nine prominent peaks read section 2 1 to learn how you can correct the problem 4 Once you are satisfied that the correct peak arrival times are in the table click the Accept button to complete the analysis 5 The active window will switch back to the Experimental Estimates worksheet after the
8. general experimental protocol detailed in Section 1 5 but augment those directions with the plate preparation and data analysis procedure described below Plate Preparation Prepare a 384 well microtiter plate as illustrated in Figure 11 for use with EZ Reader 12 sipper chips where N 70 ul of neutral dye 1 uM Bodipy Fluorescein Arginine or 150 uM Rhodamine B Sub 70 ul of 1 uM substrate peptide Prod 70 ul of 1 uM product peptide S P Sub Prod mixture 70 uL of 1 uM product and 1 uM substrate B 70 ul of 1X separation buffer Ooooo 13 14 15 16 17 Figure 11 Plate layout for using only 4 sippers of a 12 sipper chip to make viscosity mobility measurements With the plate prepared as shown in Figure 11 only sippers 7 8 9 and 10 will participate in the experiment The other sippers just sip buffer all the time This presents a small problem for the data analysis macro since it will try to analyze the data from all 12 sippers You can intervene to correct that by editing the peak arrival table You just need to delete the peak times from all of the sippers that you want to ignore leave blank entries in the table for sippers 1 2 3 4 5 6 11 and 12 Then click the Accept button amoo Caliper Life Sciences Inc 12 Optimizer for Mobility Shift Assays More about Measuring the Mobility and Viscosity Peak Finder Results Auto Mode Filename H Projects MSOptimization ValidationData CDK2 CDK2_VISC
9. the optimal separation conditions of voltage pressure and buffer sip time for a Mobility Shift Assay The Optimizer uses a mathematical model of the electrokinetic and pressure driven transport in the LabChip microfluidic devices to predict the resolution of product and substrate peaks for any applied voltage and pressure You will use the follow steps to determine the best separation conditions Install the Optimizer spreadsheet Open the Optimizer_Template Excel workbook Enter the amino acid sequence of your peptides Generate the product peptide Perform the parameter measuring experiment to measure the peptide mobility electroosmotic flow and buffer viscosity Use the Optimizer spreadsheet to determine the best separation conditions pressure voltage and sip times required to achieve your desired throughput data quality and unattended run time 7 Test the separation conditions OV ONS D Each step is described in more detail in the following section 2 Step By Step Guide to Using the Optimizer Spreadsheet 2 1 Install the Program To use the Optimizer Spreadsheet you must have a computer with Windows XP Windows Vista or Windows 7 and Microsoft Excel 2003 or higher Security Level Medium or Low installed Install the Optimizer Spreadsheet by running Setup exe from the installation disk Most of the program resides in a Microsoft Excel Add in module that will be loaded and configured by the installer See th
10. 0 83 40 107 80 272E 06 2 09E 06 1 02E 04 1 00E 04 6 87E 07 1 38E 06 1 38 1 00 2 84 90 85 10 85 10 84 70 85 10 73 90 84 60 84 30 109 70 268E 06 2 06E 06 1 03E 04 1 01E 04 1 35E 06 202E 06 1 39 1 00 3 84 10 84 40 84 30 84 10 84 50 73 50 84 00 83 70 108 60 2 70E 06 2 07E 06 1 02E 04 1 01E 04 9 36E 11 6 83E 07 1 38 1 00 4 83 90 84 20 84 00 83 80 84 30 73 20 83 80 83 40 108 10 340E 06 279E 06 1 03E 04 1 00E 04 6 87E 07 687E 07 1 38 1 00 5 6 T 8 9 10 11 12 Mean 84 20 84 45 84 40 84 10 84 52 73 48 84 03 83 70 108 55 2 87E 06 2 256 06 1 02E 04 1 01E 04 6 80E 07 1 19E 06 1 38 1 00 Results Output Estimated Substrate Product EO Electrical Substrate Product Mean Observed Observed Observed Observed Observed Observed Viscosity Mobility Mobility Mobility Resistance Retention Retention Current Current1 Current2 Current3 Current4 Current5 Current6 cP _ cm V s _ cm V s _ cm V s MO Factor Factor pA pA vA pA pA pA uA 138 2566 06 1026 04 256607 20103 100 100 6 96 6 94 6 99 Summary Electro Mobilitas c a Retention Factors must TO o 1st V 287E A TE OE 6 80E 07 be less than 1 03 2nd V 2 25E 06 1 01E 04 1 19E 06 Mean 2 56E 06 1 02E 04 2 56E 07 Difference 6 211E 0 06 06 q Mobility must be different for substrate and product Figure 13 Typical values measured in a Viscosity Mobility experiment as shown on the Experimental Estimates worksheet Close inspection of the results may reveal prob
11. 0 ul of 1 uM product peptide in separation buffer S P Substrate Product mixture 70 uL of 1 uM product and 1 uM substrate The S P wells will be sipped to test the separation using the Make an Experiment Job File function see Section 1 7 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 L N Sub Prod S P _N Sub ProgfS P _ T T T T JT J JT Lr T T T T C T T T J JT Figure 2 Plate layout for use with 4 sipper chips 4 5 6 A N N N N NIN Sub Sub Sub Sub Sub Sub UT 2 3 4 5 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 ae ae ee ee ee a T OA S T o T o T T T S T T T Figure 3 Plate layout for use with 12 sipper chips 3 Prepare the instrument and refresh the separation buffer if needed Prepare the chip and load it into the instrument Place the plate into the instrument Run the appropriate measurement job file for your chip and peptide gt The Optimizer installation CD contains several job files to be used for the measurement experiment Chip Peptide Job Filename Charge EZReader 4 Positive EZReader4 sipper_Pos_ViscosityMobilityEstimate EZReader 4 Negative EZReader4 sipper_Neg_ViscosityMobilityEstimate EZReader 12 Positive EZReader12 sipper_Pos_ViscosityMobilityEstimate EZReader 12 Negative EZReader12 sipper_Neg_ViscosityMobilityEstimate
12. 090 P ar 1500 2048 176 282 223 3203 7396 o62 1 00 rq 1900 31 01 176 353 293 3194 7387 062 1 00 r a7 2600 6475 175 635 400 3178 7372 061 100 oso 011 Figure 19 Example of the Mobility Shift Calculator worksheet Caliper Life Sciences Inc 21 Optimizer for Mobility Shift Assays Appendix B Mobility Shift Calculator Worksheet B 1 Definition of Output Values dT The time difference between the substrate and product peaks Resolution The resolution of the substrate and product peaks as defined in Section A 1 1 Incubation Time The Incubation Time is defined as the length of time it will take for a fluid element to pass through the chip s incubation channel from input to output For an On Chip incubation assay the Incubation Time is the length of time that the enzyme substrate and sample are allowed to react before arriving at the entrance to the separation channel Note that the EZ Reader 4 and 12 sipper for the Off Chip Incubation assay do not have an incubation channel so the incubation time is reported as zero seconds Separation Time The Separation Time is defined as the average length of time it will take for a Substrate molecule to travel the length of the separation channel at the given voltage and pressure This calculation includes the effects of the pressure driven flow electroosmotic flow and electrophoretic mobility of the substrate molecules Sip toDetect T
13. 2 56E 06 1 02E 04 256E 07 201 03 1 00 1 00 6 96 6 94 6 99 Summary Electro Mobilities cm7 V s Sub Ep Prod wEp pEo 1stV 2 87E 06 1 02E 04 6 80E 07 2QndV _2 25E 06 1 01E 04 _1 19E 06 Mean 2 56E 06 1 02 04 256E 07 Difference 6 211E 07 1 628E 06 1 872E 06 Figure 4 The Experimental Estimates worksheet Use this worksheet to analyze the data generated by the Viscosity Mobility measurement experiment Caliper Life Sciences Inc 6 Optimizer for Mobility Shift Assays Step By Step Guide to Using the Optimizer Spreadsheet Peak Finder Results Auto Mode Filename H Projects MSOptimization ValidationData MAPKAPK2 WISCMOB_FS_MAPKAPK2_2003 08 20_17 39 35 RDA 0 08 0 161 0 281 0 38 0 48 0 561 0 681 0 78 0 88 0 98 1 081 1 181 1 28 1 381 1 461 1 581 Accept Review the charts and edit the table if needed then click Accept 1 132 4819 231 3819 330 8819 431 1619 528 4619 640 7819 727 7819 826 4819 919 1619 2 131 5819 230 5819 330 0819 430 2819 527 4819 640 3819 726 7619 627 7619 918 0519 3 131 7819 230 8819 330 2819 430 4619 527 7619 640 5619 727 0819 828 0819 916 2619 4 132 4819 231 4619 330 0819 431 1819 526 4819 640 6819 727 7619 628 6619 919 1819 Sipper 1 140 120 100 80 1o SSS 20 0 0 200 400 600 800 1000 1200 Time sec Figure 5 The Peak Finder Results worksheet that shows the data generated by the Viscosity Mobility measurement
14. Buffer Sip Time is a parameter that must be set in the instrument Experiment Job file Post Dye Buffer Sip Time The length of time required for the instrument to sip buffer after each row marker dye so that the row marker does not interfere with the first sample of the row This is another parameter that must be set in the instrument Experiment Job file Final Delay The length of time required for the instrument to sip buffer at the end of a plate so that the last sample sipped usually a row marker has enough time to flow through the chip past the detector and allow the signal to return to baseline before the instrument stops recording data The Final Delay is a parameter that must be set in the instrument Experiment Job File Time for One Plate Approximate time required to run one 384 well plate This calculation accounts for the Sample Sipping Time Post Sample Buffer Sipping Time Robot Movement Time Row Marker Sipping Time and the Final Delay and it adjusts the time appropriately depending on the type of chip 4 sipper 12 sipper etc Peak Order The order in which the substrate and product peaks will appear in the output data file from the Caliper HTS instrument Product First or Substrate First This information is provided to make it more convenient for the user to set the Peak Order in the data analysis software Buffer Lifetime The Buffer Lifetime is the amount of time it will take for the buffer in the electrode w
15. Caliper logo LabChip and LabChip logo are registered trademarks of Caliper Life Sciences Inc All other trademarks are the sole property of their respective owners The purchase price of the accompanying product s includes a limited license under Caliper patents to use only those product units provided herewith and does not include a license to use any other Caliper products or system components that may be covered by Caliper patents The product s is licensed for Customer s internal research use only and Customer acquires no right under Caliper patents by implication estoppel or otherwise to use the product s for any other purpose including but not limited to use for diagnostic purposes or use in a screening database business As used here screening database business means providing a third party with access to a screening database containing experimental results of interactions between a collection of potential drug molecules and more than 10 different biological targets Contact Caliper Life Sciences Customer Service at 1 877 LabChip 1 877 522 2447 or visit us on the world wide web http www caliperLS com Download Mobility Shift Assay Application Notes at http www caliperls com support reference library application notes labchip systems apps htm Caliper Life Sciences Inc 2 Optimizer for Mobility Shift Assays Introduction 1 Introduction The Optimizer for mobility shift assays is used to systematically determine
16. Electroosmotic Mobility The expected range of electroosmotic mobility uEo for a buffer containing about 0 1 Coating Reagent 3 is 1e 5 to 1e 5 cm Ves Measurements outside that range tend to indicate an error in the buffer composition or a failed experiment Double check the buffer and repeat the measurement Caliper Life Sciences Inc 13 Optimizer for Mobility Shift Assays More about Measuring the Mobility and Viscosity 4 Viscosity The range of buffer viscosity that is valid for use with the standard viscosity mobility measurement job files is 1 2 cp to 1 8 cp A buffer viscosity measurement outside of that range may indicate an error in the buffer composition or a failed experiment Double check the buffer composition and repeat the measurement If you continue to observe viscosity measurements outside of the valid range contact Caliper s Customer Support group for advice call 1 877 522 2447 If you need to work with a buffer that has a viscosity outside of the range stated above you will need to use a custom measurement job file since the timing of the voltage transitions depends on the buffer viscosity Run Auto Mode Analysis Macro Manual Mode C Projects EZ Reader 1 5 1400 1400 30 106 0 2 1 6 32 5 EZ Reader 4 Sipper Electro Mobilities cm2 V s Sub a jeas ing Pa P EA pEo 1st V ata Viscosity retin R Sipper to Detector Time s Factor 1 83 90 84 10 84 20 83 80 84 20 73 30 83 7
17. Life Sciences Inc 18 Optimizer for Mobility Shift Assays Appendix A Mobility Shift Assay Predictions Worksheet A 3 Interpreting the Input Data Table Definitions of the input data table on the Experimental Assay Predictions worksheet Chip Use the drop down menu to select the type of chip that you will use for your assay For any particular assay the best operating conditions of voltage pressure and Buffer Dwell will in general be different for each chip type Viscosity The viscosity of the assay buffer in centi poise cp The user may type in any value for a hypothetical simulation however to obtain useful predictions of operating parameters for a particular assay the viscosity must be measured using the viscosity mobility experiment described in Section 1 5 Substrate Product Electrophoretic Mobility When placed within an electric field an electrically charged solute will migrate through a buffer with an electrophoretic velocity Ugp UepE cm s Where pep is the electrophoretic mobility and E is the applied electric field Separation is achieved because the substrate and product migrate through the chip at different velocities The user may type in any value for the substrate and product electrophoretic mobility to perform a hypothetical simulation however for best results when developing an assay for a particular substrate and product peptide the electrophoretic mobility must be measured using the viscosity mobility e
18. OSITYMOBILITY_12SIP_PROFESSOR_AS591A 0372N 01_17 36 10_2 Accept Review the charts and edit the table if needed then click Accept 0 056517 int 366 1443 590 2444 924 4443 1023 944 j DES besa roguns 253 5444 366 0443 590 7444 7337444 925 0444 1024 444 and leave blank entries 2536444 365 9444 4786443 590 6443 733 9443 813 4443 925 0444 253 5444 306 8444 366 0443 4911443 590 6443 733 9443 8130444 A om 141 2444 2536444 3661443 4787444 5907444 733 9443 B13 244f 9249443 1024544 141 1444 253 6444 366 0443 4786443 590 6443 7335444 813 1943 924 8444 1024544 140 6444 253 1444 365 6443 478 2444 590 2444 7336443 812 443 924 4443 1024044 140 7444 253 2444 365 6443 478 3444 590 3444 B444 924 6443 1024 244 253 1444 365 8444 590 4443 924 7444 1024 244 12 0 056517 ON Om Wh Sipper 1 2443 34434 35 5443 4 25 NMON UO OON MH 7443 i No peaks since this 8443 sipper is just sipping 0 944394 10 buffer 1 04434 5 1 144349 0 1 2443 0 200 400 600 800 1000 1200 1400 1 3443 Ti 1 4443 ime e0 Sere SS Figure 12 Illustration of a 12 sipper chip where only 4 sippers 7 8 9 and 10 are used to measure the viscosity and mobility The user must edit the peak table by deleting the values for unused sippers before clicking the Accept button 3 3 Review the Viscosity Mobility Measurement Results Close inspection of the results obtained from the viscosity mobility measurement exp
19. W Ca li 7 Optimizer for Mobility Shift Assays User Guide Introduction Table of Contents 1 MINWOGDUCTION sisii sc genacaadesasea ce dandenacelssgucs sundes ddbunnaa cvetndaaddveugad cwsaseadlenaneadddaa pecedaway 3 2 Step By Step Guide to Using the Optimizer Spreadsheet cccccccceceeeeeeeeceeceeeeeeeeecenaeeeeeeeteeenaees 3 2 1 Install the PrOQKhAM wii2 24 sactvesies ieee avis lave area en dave aaa eane en 3 2 2 Open the Optimizer Template ssia a E A A aa aa 3 2 3 Enter the Amino Acid Sequence of your Peptide ccccececeeccececeeeeeeeeneeeeeeeeeseceneaeeeeeeeseeenaees 3 2 4 Generate the Peptide Product ccccccceecececceceeeeeeeeeeeeeceeeeeeeseceanaeceeeeeseceneaneeseeeeeeatenneeeeeeeeteees 4 2 5 Perform the Viscosity Mobility Measurement Experiment ceeceeeeeeeeeeeeeeeneeeeeeaeeeeeeneeeeeeaas 4 2 6 Analyze the Dalla crensian nni cei E edie neilelitenedl E 6 2 7 Select the Best Operating Parameters cc ccccceecccceeeeeceeeeeeeeceeeneeeeeesneeaeeeneeaeeeeneeceeeeneneeenenanes 7 2 8 Test the Separation Conditions cccccccccceeeeeeccce cece ee eeeeeeeececeeeeeseceanaeeeeeeeeesegeaeaeeeeeeeeesesnanaeeeees 8 3 More about Measuring the Mobility and Viscosity 0 0 0 ee eeeeeeeeeeeee ee eeeeeeeeeeeeeeeeseeeeeeeseeeaeeeseeeaeeeeeenaees 9 3 1 Troubleshooting Analysis of the Data eccccecceeeeeeeeeeeeeeeeeeeeseneeeeeseeeeeeeseeeeeeeseneeeeeseeaeeesennaees 10
20. arameters e g Voltage Pressure sipping time etc set appropriately to achieve good performance for this particular assay Caliper Life Sciences Inc 7 Optimizer for Mobility Shift Assays Step By Step Guide to Using the Optimizer Spreadsheet Off Chip Mobility Shift Assay Voltage Pressure Predictions Y Caliper d ifeSciences Jse this page to predict Voltage Pressure pairs that will produce your desired resolution his worksheet takes its input from the Experimental Estimates sheet Use this sheet for best results when you have measured the peptide mobility Ez Reader S 7 4138 2 56E 06 1 02E 04 2 56E 07 1 17E 06 1 15E 06 1 8 16 0 2 Make an Experiment Job File Outputs Applied Post Post Dye ol Peak Voltage V Sample Bufer e Miss A Ba aol Order e Oat WAWAWIAIAIAAIAAAAAAAAAGAAAAAAA AAS Figure 6 A Typical Mobility Shift Assay Prediction worksheet Click the radio button at the left to select the set of parameters that will give acceptable throughput Time for One Plate and Buffer Lifetime and then click Make an Experiment Job File 2 8 Test the Separation Conditions This step uses the mixture of product and substrate that was put on the plate during the measurement experiment setup S P in Figures 2 and 3 to test the separation conditions 1 At the Caliper instrument open the Job or Experiment file that was created in the previous step 2 Set the Data Location and file na
21. e Optimizer Release Notes for more details 2 2 Open the Optimizer_Template On the Windows Start menu select Programs gt Caliper Life Sciences gt Optimizer Microsoft Excel starts and a new workbook is created based on the Optimizer Template It is best to start with a new instance of the template workbook for each new assay and or measurement rather than reusing a workbook that has been used before It is especially important to avoid reusing a workbook that has been modified 2 3 Enter the Amino Acid Sequence of your Peptide In the Microsoft Excel spreadsheet select the A Priori Estimates worksheet and describe the substrate and product peptides by entering their amino acid sequence and termination method in the appropriate cells see Figure 1 To enter the sequence use the single letter abbreviation for each amino acid To enter a phosphate enclose PO3 in parentheses PO3 as shown in the illustration in Figure 1 Caliper Life Sciences Inc 3 Optimizer for Mobility Shift Assays Step By Step Guide to Using the Optimizer Spreadsheet 5FAM LRRASLG CONH2 5FAM LRRAS po3 LG CONH2 Substrate Product Sub Prod Sub Prod gare ae goer ae Mobility Mobility Diffusivity Diffusivity g mol g mol cm V s cm V s cm2 s cm2 s 1130 0 11 4209 432 135 5 572E 06 6 258E 05 1172E 06 1 146E 06 Recommended Measurement Job File xxxxx_Neg_MobilityViscosityEstimate Job Where xxxxx is the chip number fo
22. ell to change by 1 pH due to the electric current passing through it see Figure 18 The function implemented in the spreadsheet estimates the time for the upstream electrode and the downstream electrode then returns whichever value is smaller The calculation assumes that the electrode wells on the chip have been filled with the recommended volume of buffer Be sure to fill the wells correctly to achieve the predicted buffer lifetime Caliper Life Sciences Inc 20 Optimizer for Mobility Shift Assays Appendix B Mobility Shift Calculator Worksheet pH of Upstream Electrode pH of Downstream Electrode 11 10 pH o 0 5 10 15 20 25 30 35 Time Hr Figure 18 Illustration of the change in pH of the Upstream and Downstream electrode wells over time for an EZ Reader 12 sipper chip with 100mM HEPES buffer The Buffer Lifetime is defined as the amount of time it will take for the pH to change by 1pH unit about 10 hours in this example Appendix B Mobility Shift Calculator Worksheet The Mobility Shift Calculator worksheet is provided as an aid to perform more detailed analysis of an assay if you wish The calculator allows you to input any arbitrary set of input parameters like viscosity mobility voltage pressure etc and it will predict the expected peak separation resolution concentration factors etc Our intent is to allow the user to customize this works
23. eriment may reveal some common problems with the assay or buffer A few things to look for are 1 Retention Factor Check to be sure the Substrate Retention Factor and Product Retention Factor are less than 1 03 The retention factor is the ratio of arrival time of the dye to arrival time of the substrate or product It is a measure of the stickiness of the substrate product relative to the dye We assume the dye is not sticky so a retention factor of 1 means the substrate or product is also not sticky If the substrate or product solution tends to stick to the glass surface of the chip the solution will move more slowly through the chip than the dye so it s retention factor will be greater than 1 A retention factor of something greater than 1 03 usually means the substrate or product is too sticky to use in a Mobility Shift assay Changing the buffer composition can solve most sticky problems See the Off Chip Mobility Shift Assay Development Guide for more details on how to solve substrate product sticking problems 2 Substrate and Product must have different mobility Sometimes the procedure used to convert the substrate into product fails so that no product is actually produced In that case the solution that you think is product is actually still just the substrate If you perform the viscosity mobility measurement with product that is really substrate the spreadsheet will say that the product and substrate have about the same mobility 3
24. experiment Be sure to click the Accept button to enter the peak arrival times into the program 2 7 Select the Best Operating Parameters 1 Switch to the Experimental Assay Predictions worksheet see Figure 6 The input cells on this sheet are all filled in by the analysis macro that was run in the previous step so you do not need to enter any values Select the set of operating parameters that best meet your needs of throughput and unattended run time Consider the columns labeled Buffer Lifetime hr and Time for One Plate min Select the set of parameters that will give adequate Buffer Lifetime without taking too long to run each plate You can select any set of parameters from the table but a good choice is to select the conditions that will give a Buffer Lifetime of about 36 hours using a pressure in the range 1 2 to 2 psi and an applied voltage lower than 2500V positive or negative To make your selection click on one of the Radio Buttons to the left of the table your selection will be highlighted in blue If there are no valid conditions available in the table for example all Impossible or if the available conditions do not meet your needs for throughput or buffer lifetime read the troubleshooting sections 2 1 and 2 3 or call Caliper Customer Service for help Click Make an Experiment Job File and enter a file name in the dialog box The spreadsheet will create an Experiment or Job file with all of the p
25. heet it s just an Excel Spreadsheet after all to suit their interests and needs for various different analyses For example you might use this sheet to perform a sensitivity analysis on the viscosity to see how much the resolution would change for your assay due to the change in viscosity that accompanies changing temperature in your lab Mobility Shift Calculator YP Caliper se this worksheet if you wish to do more detailed analysis of various operating conditions You Sciences an use this sheet to predict many interesting performance characteristics given any arbitrary set of input conditions Try changing any of the inputs voltage pressure viscosity mobility etc and see how the changes affect the performance of your assay Version _3 3 28 0 3 2 0 OffChi 2 56E 06 1 02E 04 2 56E 07 1 17E 06 1 15E 06 1 75 Pe ae Som ani Disp_P Total_S Total_P FWHM_S FWHM_P Peak a s Time s 300 23 478 471 034 3244 7437 17 400 310 178 169 045 3246 7440 raz 500 378 178 167 085 3248 7442 rat 600 444 178 165 065 3251 7444 063 rag 700 508 178 163 074 3253 7447 P ar s00 568 178 161 os4 3255 7449 r 47 1000 682 178 158 101 3260 7454 063 rq 1400 886 179 153 133 3269 7463 i TE 390 388 477 222 124 3216 7410 062 081 P a7 1200 14 60 176 248 172 3209 7403 062
26. ime The average length of time it will take for a Substrate molecule to travel from the tip of the sipper to the detector at the given voltage and pressure Substrate Product Concentration Factor If the concentration of substrate is 1 just before entering the separation channel the concentration will be Conc_S Conc_P at the detector A change of concentration may occur due to dilution by Electroosmotic Flow entering from the Upstream Electrode channel and or due to a change in velocity of the species as it enters the separation channel recall that velocity and concentration are inversely related by the principle of conservation of mass flux Substrate Product Dispersion Factor Disp_S Disp_P Predicts the normalized peak magnitude of a short sipped slug which has been spread by dispersion due to the pressure driven flow as the slug has travels through the chip from sipper to detector If there were no dispersion the dispersion factor would be 1 Full Width at Half Maximum The width of the substrate product peak at half of its maximum height Peak Order The order in which the substrate and product peaks will appear in the output data file when the assay is run Product First or Substrate First This will help identify which peak is the product and which peak is the substrate as observed in the data file Caliper Life Sciences Inc 22 Optimizer for Mobility Shift Assays
27. is less than 50 so that the contaminating substrate peak is taller than the product peak corrective action must be taken Perhaps the best thing to do is to make more product and repeat the measurement experiment You may also edit the peak table to manually enter the arrival time of the correct product peak as illustrated in Figure 9 Caliper Life Sciences Inc 11 Optimizer for Mobility Shift Assays More about Measuring the Mobility and Viscosity VISCMOB_CKE2_2003 08 14_16 33 35 RDA CCD2 1 Extra peak due to incomplete product formation f en Ma 100 20 200 aw s 60 70 200 00 4000 Time Seconds Figure 10 Example of double peak in the viscosity mobility measurement data due to incomplete product conversion If the contamination of the product by the substrate is small it can be ignored If the substrate part of the peak is greater than the product peak corrective action needs to be taken 3 2 Example using only 4 sippers of a 12 sipper chip The following example is provided to further illustrate the technique of editing the peak arrival table before clicking the Accept button and also to provide a practical means of saving reagent costs during the assay development process As a means of saving reagent costs it is possible to perform the viscosity mobility measurement experiment using only four of the twelve sippers on an EZ Reader 12 sipper chip To do this follow the
28. lems with the measurement or the buffer or reagent preparation Caliper Life Sciences Inc 14 Optimizer for Mobility Shift Assays Appendix A Mobility Shift Assay Predictions Worksheet A 1 Selecting the Resolution In order to balance data quality and throughput Caliper recommends e Foran Off Chip Incubation assay set Resolution 1 5 and Resolution Between Wells gt 2 0 Where the Resolution refers to the substrate and product peaks and the Resolution Between Wells refers to the resolution of the lagging peak of one well to the leading peak of the next well as illustrated in Figure 14 Sep_Check CA LC3000 2K 3_2007 02 08_15 26 35 rda p a ccbD2 1 ccbD2 2 ccb2 3 ccD2 4 ccbD2 5 ccD2 6 CCD2 7 ccb2 8 ccbD2 9 ccbD2 10 ccb2 11 ccbD2 12 2000 i E Resolution 1 5 it i s i I 1 i H g l o 1000 i i f i i i Ti q y 1 ll g Resolution ik i 4 i ae E i 1 so Between Wells 2 0 lz NY BA A i i N lt Ya 4 id 5 so s 125 150 175 s Figure 14 Example Mobility Shift data illustrating the characteristics of Resolution 1 5 and Resolution Between Wells 2 0 30 0 fi 1 1 i i 1 1 0 50 100 150 200 250 300 350 400 450 500 60 R 1 2 40 20 4 0 i fl I I i I I 0 50 100 150 200 250 300 350 400 450 500 40 T T T T T 0 1 fi l 1 l I I 0 50 100 150 200 250 300 350 400 450 500 Time s Figure 15
29. lution will impact the throughput and iv given the limits of performance of the instrument it may not always be possible to achieve the resolution that you desire Let s consider some of these topics in more detail e Plan for Variability in the Resolution If the desired resolution is set to some value R and the recommended voltage and pressure are used it is likely that the resolution actually achieved when the assay is performed will have some error or variation about R The main sources of error are i Variation in temperature which will change the buffer viscosity ii Preparation of the reagents iii Change in pH of the buffer over time iv Normal manufacturing tolerances of the chips and v Measurement error when measuring the viscosity and mobility of the reagents The assay developer should anticipate 10 to 20 variation in the resolution depending on the degree of control that is placed on the sources of error To make a robust assay that is insensitive to changes in the variables listed above it is best to factor in some margin of safety when selecting the resolution The assay developers at Caliper follow the convention of setting the Desired Resolution 1 5 and the Resolution Between Wells gt 2 0 These values have been chosen since the lower limit on the resolution that Caliper s data analysis software can reliably process is R 1 2 and we want to allow for a 20 worst case variation in the resolution from day to day d
30. ming convention as appropriate for your application and save the file 3 With the same plate previously used for the measurement experiment on the plate tray start the run You can skip the preparation steps in the start wizard or Setup wizard on EZ Reader instruments since you have already completed them during the first part of the experiment 4 Visually inspect the resulting data file to be sure that the Resolution between substrate and product peaks and Between Well Resolution are acceptable see example in Figure 7 Caliper Life Sciences Inc 8 Optimizer for Mobility Shift Assays More about Measuring the Mobility and Viscosity 5 Assuming the Separation Check data is acceptable you can use the Separation Check file as a template for your screening job or experiment just modify the Dwell Pattern Data Location etc and save it with a different name If the Separation Check data file looks unacceptably different than the example in Figure 7 read Section A 2 Troubleshooting Resolution Errors Sep_Check_CA LC3000 2K 3_2007 02 08_15 26 35 rda ccD2 1 ccD2 2 ccbD2 3 ccD2 4 ccD2 5 ccD2 6 N CCD2 7 cCD2 8 ccb2 9 CcCcbD2 10 cCcbD2 11 ccbD2 12 2000 Resolution 1 5 1000 CResolution so Between Wells 2 0 Figure 7 Example of a good Separation Check file illustrating the two most important characteristics to check Resolution 1 5 and Resolu
31. persion of the peaks and to a lesser extent due to small differences in the resolution of the peaks If you produce a Separation Check file that is unacceptably different than the examples shown here follow the steps listed below to try to correct the problem Caliper Life Sciences Inc 17 Optimizer for Mobility Shift Assays Appendix A Mobility Shift Assay Predictions Worksheet 40 20 o m 50 100 150 200 250 oO 40 20 oO a Oo 100 150 200 250 300 350 400 400 200 oO 50 100 150 200 250 300 Time sec Figure 17 Examples of acceptable Separation Check files for several different assays a Fyn b CK2 c PRAK The assays each have different shaped peaks due to differences in the mass and charge of the peptides used Even though they look somewhat different they are all considered acceptable and the assays have given good performance What if do not actually achieve the resolution that the spreadsheet predicts The most likely reason for errors in the predicted resolution is a poor estimate of the viscosity or mobility Try some of the following things to improve the measurements 1 Double check the Peak Finder Results worksheet looking for anomalies like extra peaks in the data signal that might have interfered with the analysis of the data see Section 2 1 and Figure 9 2 Review the viscosity mobility measurement results on the Experimental Estimates worksheet as described in Section 2 3 and
32. r example EZ Reader 4 Sipper or EZ Reader 12 Sipper Figure 1 The A Priori Estimate worksheet The substrate and product peptides must be described by entering their amino acid sequence and termination into the appropriate cells on this sheet This worksheet calculates a molecular diffusivity estimate that is used by other worksheets to predict assay performance The output values for the electrophoretic mobility should be treated as ballpark estimates based simply on the mass and charge of the molecules They are displayed for your information only for best results the electrophoretic mobility must be measured in the assay buffer that you intend to use for screening Use the Recommended Measurement Job File to measure the mobility as describe in the next section 2 4 Generate the Peptide Product Purchase a phosphorylated peptide or use an enzymatic reaction to generate pure product peptide About 70 product is sufficient for reliable measurement measurement errors may result if the product is contaminated by more than about 30 with the substrate peptide 2 5 Perform the Viscosity Mobility Measurement Experiment It is important to perform the viscosity mobility measurement experiment using your particular assay buffer Caliper s recommended buffer Caliper 760367 P is described below as a guide If your assay requires any changes to the buffer be sure to make the changes when performing the viscosity mobility measurement
33. ree buffering components needed for the buffer lifetime calculation Resistance of Separation Circuit The electrical resistance of the chip s separation circuit The resistance is a function of the chip geometry as well as the conductivity of the buffer This value is measured as part of the viscosity mobility measurement experiment Robot Movement Time This is the Travel Time stated in the RDA file divided by 2 since the RDA file states the round trip robot travel time The Robot Movement Time is measured as part of the viscosity mobility measurement experiment It is typically about 1 5 seconds Sample Sip Time The Sample Dwell time as set in the instrument Experiment Job file typically 0 2 sec for a Mobility Shift assay Caliper Life Sciences Inc 19 Optimizer for Mobility Shift Assays Appendix A Mobility Shift Assay Predictions Worksheet A 4 Interpreting the Output Data Table Definitions of the output data table on the Experimental Assay Predictions worksheet Voltage The voltage values listed in the output table are the difference of potential to be applied between the upstream electrode well and the downstream electrode well Where the positive voltage is defined as the one that will propel a positive charge from the entrance of the separation channel toward the detector Therefore with a positive voltage applied the upstream electrode will be positive with respect to the downstream electrode If
34. stimate experiment described in Section 1 5 Electroosmotic mobility When an electric field is applied across the LabChip s separation channel the buffer will be set in motion with a velocity Ugo peoE cm s Where peo is the electroosmotic mobility and E is the applied electric field The user may type in any value for a hypothetical simulation however to obtain useful predictions of operating parameters for a particular assay the electroosmotic mobility must be measured using the viscosity mobility experiment described in Section 1 5 Substrate Product Molecular Diffusivity The molecular diffusivity coefficient is used by the spreadsheet to predict the amount of spreading or widening the sipped samples will experience as they pass through the chip Short peptides typically have a molecular diffusivity value of about 1x1 0 cm s The user may type in any value for the substrate and product molecular diffusivity however for best results when developing an assay for a particular substrate and product peptide use the A Priori Estimates worksheet to estimate the values based on the amino acid sequence as described in Section 1 3 pH pH of the buffer This information is needed for the buffer lifetime calculation Buffer1 2 3 Concentration of buffering species needed for the buffer lifetime calculation The user may provide values for up to three buffering components in the buffer pK1 2 3 pK values for up to th
35. the desired resolution cannot be achieved by applying a voltage within its search range the function that solves for the voltage returns Impossible meaning it is impossible to achieve to desired resolution The function that solves for the voltage searches within the range of 200V to POSITIVE_VMAX and 200V to NEGATIVE_VMAX Where POSITIVE_VMAX is equal to the highest voltage that can be applied without producing a total loss of the charged species to the Upstream Electrode or ABSOLUTE_VMAX whichever is smaller NEGATIVE_VMAX is equal to the largest negative voltage that can be applied without producing a total loss of the charged species to the Upstream Electrode or ABSOLUTE_VMAX whichever has the smaller magnitude Where ABSOLUTE_VMAX is the maximum value that can be produced by the power supply By limiting the search range in this way the spreadsheet avoids recommending a voltage that is outside the limits of performance of the instrument or that would produce a loss of the substrate or product to the upstream electrode well Post Sample Buffer Sip Time The length of time required for the instrument to sip buffer between each sample to produce desired Resolution Between Wells The Resolution Between Wells is defined as the resolution of the lagging peak of one well to the leading peak of the next well You need to consider the Resolution Between Wells to ensure that adjacent samples will not interfere with each other The Post Sample
36. time of one or more of the peaks After the automatic peak finder has completed processing the data file the user is asked to review the plot for each sipper to verify that the nine correct peaks have been found and then click the Accept button if that is true Most of the time there are only nine prominent peaks in the file and it is obvious that the correct ones have been identified However Figure 9 shows an example where a few extra peaks are present probably due to some contamination of reagents and one of the extra peaks labeled in the figure is more prominent than the neutral dye peak that the analysis program is actually trying to find In the example the analysis program has incorrectly identified the Peak as the dye peak and it would make a large measurement error if it were not corrected Perhaps the best thing to do in a case like this is to identify the source of the supposed contamination correct that problem and repeat the measurement experiment with fresh reagents In the event that some extra peak s cannot be removed from the experimental results the spreadsheet allows you to edit the peak arrival times in the Peak Finder Results table before clicking the Accept button Contact Caliper s Customer Service group if you need additional help call 1 877 522 2447 Caliper Life Sciences Inc 10 Optimizer for Mobility Shift Assays More about Measuring the Mobility and Viscosity Peak Finder
37. tion Between Wells 2 0 3 More about Measuring the Mobility and Viscosity To obtain useful predictions from the spreadsheet it is essential to have good estimates of the following 1 Viscosity of the assay buffer 2 Electrophoretic mobility of the substrate peptide 3 Electrophoretic mobility of the product peptide and 4 Electroosmotic mobility of the chip buffer system The following sections describe the measurement experiment in more detail and offers advice for troubleshooting measurement problem and analyzing some of the more complicated data files that may come about The general idea of the experiment is to sip a neutral dye e g BFA Rhodamine B and then sip a sample of substrate then product with first no voltage applied then with a negative voltage applied and finally with a positive voltage applied as illustrated in Figure 8 Caliper Life Sciences Inc 9 Optimizer for Mobility Shift Assays More about Measuring the Mobility and Viscosity FYN_8_VISCMOB_A576B 0266N 02_ELMERFUDD_2003 09 24_16 53 14 RDA Current2 CCD2 1 C 40 V 1400V V 1400V o 100 200 300 400 500 600 700 800 900 1000 1100 Time Seconds Figure 8 Typical data generated by the Viscosity Mobility measurement experiment annotated to clarify the origin of each peak and show the timing of the voltage transitions The viscosity of the buffer is measured by using the transit time from sipper to detector of the neu
38. tral dye and knowledge of the applied pressure and chip geometry to solve for the viscosity required to produce the observed transit time The mobilities are similarly estimated by using the neutral dye as a reference and comparing its travel time with the travel time of the substrate and the product while a voltage is applied Any difference in observed transit time between the neutral dye and substrate or product is assumed to be due to different electrophoretic mobilities of the molecules Since the electrophoretic mobility of the neutral dye is known to be zero it is used as a reference to estimate the mobilities of the other molecules 3 1 Troubleshooting Analysis of the Data The Optimizer spreadsheet contains all the functions and look up tables needed to analyze the data from the viscosity mobility measurement experiment The analysis is performed in two steps First the raw data file is processed to extract the time of occurrence of each of the nine peaks shown in Figure 8 Next the peak arrival times are transferred into the Experimental Estimates worksheet and the calculations of viscosity mobility etc are performed In most cases the analysis macro yields results without any intervention from the user However sometimes the measurement experiment produces a data file that cannot be analyzed automatically In this case the spreadsheet allows the user to override the automatic peak finding by typing in the correct value for the arrival
39. ue to the combination of all error sources By setting the desired Resolution 1 5 and the Resolution Between Wells gt 2 0 we allow the resolution to vary a little bit from day to day without affecting the results obtained from the data analysis software e Choice of Resolution will Affect Throughput Since the substrate and product are spatially separated inside the chip we are required to sip buffer for some period of time between each sample to ensure that adjacent samples do not interfere with each other If the resolution of the substrate and product is small their peaks will be close together so the required inter sample buffer sipping time can be small However if the resolution is large the substrate and product will be farther apart so the required buffer sipping time will be larger In short the price that must be paid for higher resolution is lower throughput so it usually does not make sense to run an assay at a resolution greater than 1 5 unless you expect more than about 20 variation from run to run A 2 Troubleshooting Resolution Errors Figure 17 shows several examples of Separation Check files produced by different assays i e different peptides Although the plots look somewhat different from each other they are all considered acceptable and the assays have all been used in successful screens The differences in the plots are mainly due to the different mass and charge of the peptides which causes more or less dis
Download Pdf Manuals
Related Search