MST Starting Guide – Monolith NT.115 Contents
Contents
1. VNC VOD amp T _ MreocrR technologies MST Starting Guide Monolith NT 115 Contents 1 How to design an experiment 2 Before you start 3 Assay Setup Pretests 4 Assay Setup 5 MST experiment using temperature control 6 Data Interpretation www nanotemper de 1 How to design an Experiment www nanotemper de VNC Ve TEMPER technologies The Monolith Systems measure equilibrium binding constants between varieties of molecules with almost no restriction to molecular size or mass Although the system is easy to handle you should follow this guide when using the instrument for the first time This guide is designed to help you to get reliable results as quick as possible More detailed information is available in the Monolith NT115 User Manual Flow Chart Assay Setu Step 1 Fluorescence Check Step 2 Capillary Check VN Ve TEMPER technologies Fluorescence check with calibration curve Fluorescence signal Fluorescence signal too low too high Control labeling efficiency and potential loss of material control free dye Sample test in standard hydrophilic and hydrophobic treated capillaries Use hydrophilic or hydrophobic treated capillaries depends on reproduceability sample sticks to standard but not to hydrophilic hydrophobic treated capillaries sample sticks in all S ICh sample does not capillaries stick at all Improve buf2. LED Power or concentration of labeled molecule Fluorescence 29 2 29 6 30 30 4 30 8 pe 31 2 31 6 Position mm Figure 3 3 Fluorescence intensity is optimal between 200 and 1500 counts How much of the fluorescently labeled molecule should use 1 Choose your labeled sample concentration in the assay according to the following criteria It should be on the order of the expected KD or less In a typical experiment 5 100 nM of the fluorescently labeled molecule are used Do not work at lt 200 fluorescence counts Note Itis important to measure at fluorescence intensities that are well above the background of the signal you get from a buffer filled capillary i e without dye 2 Fill the sample in a NanoTemper Standard Treated Capillary K002 and start a Capillary Scan with 50 LED Power with the LED that matches your dye 3 Compare the intensity to the dye calibration curve you prepared previously Note If the fluorescence intensities do not match by a factor of 2 3 then either labeling efficiency is low or protein sample concentration is not in the expected range It is not necessary to have a labeling ratio of 1 1 typically 0 5 to 1 1 is used but a very low labeling efficiency might also indicate a problem with protein activity Too much fluorescence might indicate over labeling or presence of free dye 4 Never work at less than 200 fluorescence counts Never perform MST Experiments if the fluore
3. labeled molecule Caution If the fluorescence counts of your sample deviates more than a factor of two or three from the calibration curve you either have to optimize labeling efficiency or you have less sample material than you thought Also rule out that there is free dye in the sample Characterize your labeling efficiency and sample concentration as good as possible before you proceed with assay development A labeling ratio of 1 1 is optimal but not necessary A strong deviation from the expected fluorescence might indicate a general problem of the sample Note You can test the degree of labeling by measuring the absorbance of the dye and of your protein 280 nm using a photometer 6 www nanotemper de Figure 3 1 Fluorescence signal too low Increase LED Power or concentration of labeled molecule Figure 3 2 Fluorescence signal too high Decrease YN YD amp T _ MreocrR technologies 3 Assay Setup Pretests Before you start you have to be sure that you are using the optimal concentration of the labeled molecule the correct capillary type and a buffer in which your sample is homogeneous 3 1 Fluorescence Check g K gt w Fluorescence N N y HY N a o N Ga n gt epore peepee peer peer e e eee e 72 6 72 9 73 2 73 5 73 8 741 74 4 74 7 75 Position mm Fluorescence es amp S D o So o 72 6 72 9 73 2 73 5 74 4 14 7 15 73 8 141 Position mm
4. learn if laser power has an effect on the KD you determined Always choose the lowest possible laser power for you analysis which give in a good signal to noise ratio 13 www nanotemper de
5. a protein with a fluorescent dye the protein preparation you are using has to be as pure as possible For the same reason avoid the use of carrier proteins as BSA in the protein stock that you want to label Other proteins that get labeled as well will reduce your signal to noise ratio as the free dye does Always spin down the stocks of labeled and unlabeled molecules for 5min at 13 000 rpm in a table top centrifuge This step will remove big aggregates which is one of the main sources for noise Always test the quality of your labeling procedure before you start 1 Prepare a dye calibration curve It is best to prepare your own calibration curve for NanoTemper and other dyes on your instrument e g 200nM 100nM 50nM 25nM 12 5nM 6 25nM 3 12nM 1 56nM Use your interaction buffer You should use 50 LED Power for measuring Fill the samples in standard capillaries K002 and start a capillary scan and a measurement using 50 LED Power Plot or note the fluorescence intensity in the peak of each capillary 2 Determine the concentration of your labeled molecule Prepare a 100ul dilution of 50 nM of the labeled molecule in your interaction buffer and fill it into a single capillary and place it on the tray Insert the tray in the instrument and start a scan at the capillary position at 50 LED power of the suited LED Use the fluorescence value from the dye calibration curve step 1 in order to estimate the concentration of your
6. an add different additives to the buffer Choose the buffer which gives the best signal to noise ratio IMPORTANT Samples that have the inherent property to aggregate or that show only small thermophoretig amplitudes should be tested in enhanced gradient capillaries as well www nanotemper de 4 Assay Setup A Prepare a serial titration of the unlabeled molecule Highest concentration unlabeled molecule 20 pl Add 10 ul buffer to 15 reaction tubes Transfer 10 ul from vial 1 to vial 2 mix well Transfer 10 yl from vial 2 to vial 3 mix well Repeat this steps in all vials Make a stock of the diluted label molecule 16x 10 pl and add 10 ul to each tube of the titrated unlabeled binding partner Load samples into MST glass capillaries and start MST Analysis Figure 6 A schematic overview how to prepare a MST Experiment VNC Ve TEMPER technologies Now you are ready to start your interaction experiment You have a stable sample that has a low base line noise which will allow you to detect even minute changes of your molecule of interest 1 Prepare 16 small micro reaction tubes best suited are tubes with a volume of 200ul or less Label them from 1 through 16 2 Fill at least 20ul of the highest concentration you intend to use in the first micro reaction tube number 1 3 Fill 10ul of the buffer from step B 3 into the micro reaction tubes 2 to 16 Note Avoid any buffer dilutio
7. any unspecific Sticking to the glass surfaces Depending on the type of polymer used for the coating the capillaries are referred to as either hydrophilic J K004 or hydrophobic K003 For more information visit our homepage www nanotemper de NanoTemper also offers an Assay Development Kit which contains all important capillary types Fluorescence Figure 4 1 No Sticking symmetrical fluorescence peak You can use this capillary To test the best capillary type please follow the following steps f MST E i type for your MST Experiment 1 Prepare 120ul of the labeled molecule at the concentration you want to use in the assay as determined in step 2 and 3 2 Fill 4 standard treated capillaries K002 4 hydrophilic capillaries K004 and 4 hydrophobic capillaries K003 with the same sample taken directly from the 120ul stock 3 Put the 12 capillaries on the tray insert it into the instrument and start a capillary scan using the LED settings determined in step A Please note that the capillary scan starts at the back of the tray position 16 or 12 respectively Take this into Fluorescence Figure 4 2 Slight Sticking shoulders in account when you choose the type of capillary for your fluorescence peak Please note that it might take a experiment The following graphs show examples of stable 2 3 minutes to observe a sticking effect since itis a and sticking samples diffusion limited process If yo
8. by inexperienced users or for assay setup In these cases measure with the temperature control turned off The affinity will be determined at ambient temperature of your laboratory When you want to work with a temperature controlled samples we strongly recommend you to do the following experiment before you do the actual binding experiment 1 Prepare a solution of the fluorescently labeled molecule for 16 capillaries Prepare it in the concentration you intend to use in the assay i e as determined previously 2 Turn the temperature control of the instrument on and adjust it to the respective temperature you want to use 3 Fill 16 capillaries with the same sample start a capillary scan and wait for at least 5 minutes before you start the measurement This is the time it takes for the tray and capillaries to equilibrate to the new temperature 4 Measure at 40 IR Laserpower with 30 seconds Laser on time 5 Analyze the data with the Thermophoresis and T Jump settings and look for a potential base line drift effect see Figure 7 2 11 www nanotemper de VNC VOD amp T _ MreocrR technologies 6 Data interpretation 6 1 Fluorescence Evaluation Points ne 22 oo e kd o o e gt INIRE ale hc IONE cae 10 10 10 10 1 Concentration o Figure 8 the fluorescence intensity should not vary more as 10 between different capillaries This section giv
9. epared before Fill at least 4 capillaries with sample in your binding buffer and 4 capillaries with sample in MST optimized buffer 3 Perform the capillary scan with the predetermined settings and measure the samples at 40 IR Laser Power with an on time of 30 seconds Load the results in the analysis software select the normalized fluorescence tab for time traces and the Thermophoresis with T Jump tab for analysis The data should have an average minimum to maximum noise of 4 units or less IMPORTANT If the noise is more than 8 units we strongly recommend testing different buffers to improve the result see figure 5 1 and 5 2 As a rule of thumb when a decimal appears on the left axis of the Thermophoresis with T Jump plot the quality of the Sample is good Note In many cases detergents e g 0 05 Tween 20 strongly improve the homogeneity of the sample you can also add BSA casein or reductive agents to your assay buffer It is a good practice to centrifuge solutions of conjugates in a microcentrifuge for 5 min at 13000 rom before use only the supernatant should then be used in the experiment This step will remove any aggregates that may have formed during storage Note Standard buffer recommendation MST Optimized Buffer 50mM Tris pH 7 4 150MM NaCl 10MM MgCl2 0 05 Tween 20 If no improvement could be observed using this buffer please test different buffers as Hepes Tris or Phosphate buffers You c
10. es you some hints to access the quality of your MST data Please refer to the User guide manual in order to learn more how to use the MST analysis software This is the first parameter of your results that you should check Typically the intensity should only vary by about 10 If there are stronger random variations either the mixing of the sample has to be optimized or labeled sample is lost during sample preparation pipetting micro reaction tube and the like One way to test this is using Tween 20 or BSA in the buffer If it increases counts and or stabilizes the variations then the loss of material was an issue The intensity should in general be constant within 10 throughout the whole serial dilution If there is a concentration dependent shift in intensity i e constant not random increase or decrease there are some reasons for a fluorescence change that you should be aware of e Change in fluorescence yield upon binding The electrostatic surrounding of the dye molecule changes upon binding and the intensity changes typically weak changes typically not more as 2 3 Fold change Note No need to improve the assay e Either the bound or unbound state is lost during sample preparation which leads to a concentration dependent change in fluorescence throughout the serial dilution In most case this effect may only generate a false positive response at very low fluorescence counts Improve by working at a constant leve
11. fer conditions by adding BSA Use standard treated capillaries detergent www nanotemper de VN Ve TEMPER technologies Step 3 Buffer Sample Quality Check Load samples in your buffer and in MST buffer gt 4 capillaries Measure at IR40 and IR on time 30s noise level gt 6 8 units noise level 1 4 units Please test other buffer systems You must spin down the samples You can start your before loading into glass MST Experiment capillaries Noise must be lt 4 units www nanotemper de VNC Ve TEMPER technologies 2 Before you start 2 1 Design of Experiment Check before the experiment if the concentration of the gt Ne of Capillanes peel i Woe ie hi l ee Te TENOR unlabeled molecule is high enough to reach a final d m aa concentration at least an order of magnitude ideally Find a Proper Concentration Distribution more above the expected dissociation constant oe mMin K am max Ko i a i Ia KD For details refer to the Concentration Finder Tool in the Monolith Software You can use this tool in order to simulate the binding event It will help you to choose an optimal concentration range of the unlabeled molecule Bound Unbound aa Ut Microscale Thermophoresis is a method that uses very Figure 1 Use the Concentration finder tool in small quantities and volumes of material The order to design your MST Experiment capillaries only need 4ul
12. l of BSA e g 0 1 0 5mg ml or Tween 20 up to 0 05 12 www nanotemper de 6 2 Thermophoresis and T Jump Evaluation Points is with Jump gt oOo t Thermophores wo wo Oo rn e 925 10 10 10 10 10 Concentration Fig 12 the MST amplitude should be significantly higher as the noi OONO leMPgerre technologies Depending on the interaction of interest Thermophoresis and Temperature Jump analysis tabs may all report the binding event individual as well as in combination Note The MST T Jump does not yield a result for every interaction It rare case it might deviate from Thermophoresis results since it is sensitive to the local surrounding of the dye E g if you have a mixture of monomers and dimers the T Jump might only report binding to monomers If more than just one setting shows a result they yield the same affinity constants The standard setting for evaluation is the tab Thermophoresis and T Jump There are different rules to access the quality of a signal e A signal should have more than 5 response units amplitude amplitude difference between bound and unbound state e The baseline noise should be at least 3 times less than the amplitude e You should always measure at 3 different laser powers and compare the results IMPORTANT Start always from the lowest IR Laser power 20 40 80 This allows you to get the best signal to noise and you
13. n effects The buffer in tube number one and the buffer in the other tubes must be the same Otherwise you get a gradient in salt DMSO glycerol or other additives This interferes with the MST measurement 4 Transfer 10ul of tube number one to tube number two and mix very well by pipetting up and down several times Note Do not vortex these small volumes since that will not mix efficiently and may lead to denatured protein 5 Repeat this 15 times and remove 10ul from tube number 16 after mixing 6 Mix 10ul of fluorescently labeled sample at double the concentration determined at step 3 1 with the 10 ul of the titrated compound and mix well by pipetting up and down several times 7 Incubate the sample at conditions of your choice before filling it into the capillaries In most cases 5 minutes incubation at room temperature are sufficient 10 www nanotemper de VN Ve amp T MPerR technologies 5 MST Experiment using Temperature Control Evaluation Points Hot Cold 0 5 10 15 20 Concentration Figure 7 1 No Baseline Drift All tray positions can be used Evaluation Points Concentration Figure 7 2 Baseline Drift Do not use capillary positions highlighted in red The NanoTemper Monolith NT 115 and Monolith NT LabelFree are equipped with a temperature controlled sample tray 20 50 C We do not recommend that the temperature control is used
14. of your sample Please follow the following rules e Never prepare less than 20 ul of sample Otherwise you increase the probability to encounter problems due to evaporation sticking of sample material to the plastic micro reaction tubes and higher pipetting errors Optional if your protein sticks to surfaces you may use detergent or low binding reaction tubes to stabilize the sample e Never prepare small volumes e g 20 ul in large micro reaction tubes e g 500 ul or more The high surface to volume ratio leads even for well behaved proteins to a surface adsorption Always use the smallest micro reaction tubes possible e g PCR tubes or low volume microwell plates You can also obtain MST tested tubes from NanoTemper Technologies www nanotemper de Fluorescence Counts 2 2 Quality of your labeling procedure N oO O O oO O O O O O S O O BLUE LED Power 50 50 100 150 Concentration NT495 BLUE nM Figure 2 Example of a calibration curve using NT 647 dye at 50 LED Power 200 VNC Ve TEMPER technologies Make sure that there is no unreacted free dye in the preparation of your labeled molecule If you are not sure about the quality of the labeled molecule preparations use NanoTemper Labeling Kits www nanotemper de Free dye molecules will strongly reduce the signal to noise ratio Use only highly pure protein samples for labeling If you intend to label
15. scence intensity is higher than 1500 counts To achieve this the sample concentration can be adjusted accordingly or the LED should be varied between 15 and 95 Note For high affinity interaction KD lt 10 nM the concentration of the molecule should be on the order of the KD or below If the KD is lower as the detection limit of the dye you are using use the lowest possible concentration of the labeled molecule in which you get 200 fluorescence counts at 95 LED Power Once your assay is established and you are familiar with the instrument you can also test the system with 100 200 fluorescence counts Note In case you have a low labeling efficiency or your molecule sticks to your plastic micro reaction tubes the fluorescent counts might be much lower than expected For a labeling efficiency of 1 1 10nM of label will give you a sufficient signal for almost any dye If your fluorescence is much lower than expected prepare a new dilution where you add 0 05 Tween 20 to the buffer If detergent increases your fluorescence counts you lost material in the plastic micro reaction tube before www nanotemper de Fluorescence N VNC Ve TEMPER technologies 3 2 Capillary Check Which MST capillary type should I use lat IMPORTANT Some molecules will stick to the surface of the capillary The resulting MST signal has a poor quality NanoTemper offers f different types of covalently coated capillaries to avoid
16. u are not sure start a 4 If the fluorescence peaks of the scan are symmetrical you capillary scan after your measurement Repeat the can use these capillaries and go on to the next step Scan after 2 3 minutes Note In the unlikely case that the sample is sticking to all types of capillaries you can also try different buffers e g containing BSA detergent Casein or other additives Adding detergent to the buffer might make a sample work even in standard treated capillaries Fluorescence Figure 4 3 the labeled molecule sticks to the capillary you are using You MUST test another capillary format or improve the buffer composition go to step 3 3 Figure 4 4 Very Strong and clear double peak behavior of a sticking sample You MUST test another capillary format or improve the buffer composition You can learn more how to find the best buffer in step 3 3 www nanotemper de VNC Ve TEMPER technologies 3 3 Sample Quality How can I find the best buffer ence Fluoresc o Hot Cold wv sw Cycles Evaluation Points 713 3 Concentration Figure 5 1 The graph shows 4 times the same sample measured with 40 IR power The sample quality is very poor The inhomogenity of sample is clearly seen by the bumpyness of the MST curves aggregation The Thermophoresis and T Jump result shows a minimum to maximum noise of 10 units It is strongly recommended to impro
17. ve sample quality before performing the binding experiment Fluoresc o Cycles Evaluation Points Concentration Figure 5 2 The graph shows 4 times the same sample measured with 40 IR power The sample quality is very good There are no bumps in the curves The time traces almost perfectly overlap bottom The Thermophoresis and T Jump result shows a minimum to maximum noise of 1 unit You are ready to start an experiment Please note that the sample in Fig 5 1 and 5 2 is the same The sample in Fig 5 2 was measured in MST optimized buffer and was spinned down at 13 000 rom for 5min Up to now you have chosen a suitable concentration of the labeled protein sample and you have tested in which capillary type your sample is stable in solution In this chapter you will learn how to find the most suited buffer for your MST Experiment The most important criteria for an optimal buffer Good reproducibility of MST results meaning time traces are well overlapping for the same sample The most straight forward test for the quality is to compare the results obtained in gt 4 capillaries filled with exactly the same sample To do so please follow the following steps 1 Prepare 100ul of the sample in your binding buffer and 100ul in MST optimized buffer 50 mM Tris HCl 150 mM NaCl 10 mM MgClo 0 05 Tween 20 2 Fill the type of capillary you determined in step 3 2 using the sample stocks pr
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