User Starting Guide for the Monolith NT.115
Contents
1. 5 Data Interpretation This section gives you some hints to access the quality of your MST data Please refer to the Monolith NT 115 User Manual in order to learn in detail how to use the NT Analysis software 5 1 Fluorescence Evaluation Points 10 10 10 10 Concentration Figure 8 The fluorescence intensity should not vary more than 10 throughout the whole serial dilution Fluorescence ee co w w vw lt w Facies hice techie ELTETE The Original Fluorescence is the first parameter of your results that you should check A Random Fluorescence Changes Typically the intensity should not vary more than 10 If there are stronger random variations either the mixing of the sample has to be optimized or the labeled sample is lost during the sample preparation pipetting micro reaction tube and the like One way to test this is adding Tween 20 or BSA to the buffer If this increases counts and or stabilizes the variations then the loss of material was an issue B Concentration Dependent Fluorescence Changes A concentration dependent change in the fluorescence intensity i e non random constant increase or decrease can be explained as follows Change in fluorescence intensity 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 Either th2. TzMPEZErR technologies User Starting Guide for the Monolith NT 115 Content 1 Experiment Design 2 Before You Start 3 Assay Setup Pretests 4 Assay Setup 5 Data Interpretation www nanotemper de 1 Experiment Design The Monolith systems measure equilibrium binding constants between a variety of molecules with almost no restriction to molecular 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 quickly as possible More detailed information is available in the Monolith NT 115 User Manual www nanotemper de VNC Va MPczr Flow Chart Assay Setup Fluorescence Check Capillary Type Check Sample sticks to standard but not to hydrophilic and hydrophobic capillaries Use hydrophilic or hydrophobic capillaries Fluorescence signal too high Control presence of free dye Fluorescence check with calibration curve Sample test in standard hydrophilic and hydrophobic capillaries sample sticks in all capillary types Improve buffer conditions by adding BSA detergent Fluorescence signal too low Control labeling efficiency and potential loss of material sample does not stick at all Use standard capillaries www nanotemper de TEMPER Buffer Composition Sample Quality Check Load
3. gt 5 o l F a25 Ze A signal should have more than 5 response units BR aee aaea amplitude amplitude difference between bound and 10 10 10 10 10 Concentration unbound state The baseline noise should be at least 3 times less than the amplitude e You should always measure with 2 different MST powers 20 40 and compare the results Figure 9 The MST amplitude should be significantly higher than the noise in the baseline and the saturation IMPORTANT It is highly recommended to perform MST measurements both with 20 and 40 MST power to get the best signal to noise ratio Always choose the lowest possible laser power for your analysis which gives a good signal to noise ratio IMPORTANT If you do not obtain any binding curve with 20 and 40 MST power you should use 60 and 80 MST power For measurements performed with 60 and 80 MST power we recommend to reduce the time settings 5s Fluo Before 15s MST On 5s Fluo After in the parameter table in the NT Control software since the thermophoresis curve approaches the steady state phase earlier compared to measurements at lower MST power Depending on the interaction of interest both analysis modes Thermophoresis and Thermophoresis with Jump may both report the binding event individual as well as in combination If more than just one setting shows a result they should yield similar affinity constants Different Kas obtained by
4. different analysis modes can be explained by Different signal to noise ratios Non homogenous mixture of sample Note The analysis modes Temperature Jump does not yield a result for every interaction The Temperature Jump result might be different from the Thermophoresis result since it is sensitive to the local surrounding of the dye e g if you have a mixture of monomers and dimers the Temperature Jump might only report binding of monomers to your molecule of interest V07_2013 11 05 13 www nanotemper de Contact NanoTemper Technologies GmbH Fl ergasse 4 81369 M nchen Germany Tel 49 0 89 4522895 0 Fax 49 0 89 4522895 60 support nanotemper de http www nanotemper de www nanotemper de
5. much fluorescence might indicate over labeling or presence of free dye Note You can test the degree of labeling by measuring the absorbance of the dye and of your protein using a photometer e g measure absorption at 280 nm for protein and at the wavelength of the dye used e g 650 nm www nanotemper de VNC Ve l eMP erre 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 composition in which your sample is homogeneous 3 1 Fluorescence Check Fluorescence yvy 72 6 72 9 73 2 73 5 73 8 74 1 74 4 74 7 75 75 3 Position mm Figure 3 1 Fluorescence signal too low Increase the LED power or concentration of labeled molecule Fluorescence 72 6 72 9 73 2 73 5 74 4 74 7 75 75 3 Position if Figure 3 2 Fluorescence signal too high Decrease the LED power or concentration of labeled molecule Fluorescence 29 2 29 6 30 30 4 308 31 31 6 32 32 4 32 8 Position mm Figure 3 3 Fluorescence intensity is optimal between 200 and 1500 counts What concentration of the fluorescently labeled molecule should use 1 Choose the concentration of your labeled sample according to the following criteria It should be lower or in the order of the expected Kg In a typical experiment 5 100 nM of the fluorescently labeled molecule are used Do not work with less than 200 fluor
6. same sample in your assay buffer and in MST buffer gt 4 capillaries each and measure with 40 MST power Noise level more Noise level less than 6 8 units than 6 8 units Test other buffers and spin down You can start your samples before MST experiment usage Noise level is defined in chapter 3 3 5 www nanotemper de VNC Va MPczr 2 Before You Start 2 1 Design of Experiment Check before you start the experiment if the R TR Woe concentration of the unlabeled molecule is high era re Im rer enough to reach a final concentration at least an EEE order of magnitude ideally more above the Tr tw expected dissociation constant K For details refer to the Concentration Finder tool in the NT Analysis 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 a 10 Concentration Microscale Thermophoresis MST is a method that Figure 1 Use the Concentration Finder tool in uses very small quantities and volumes of material order to design your MST experiments Only 4 ul of your sample are needed to fill a capillary However to ensure optimal results 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 re
7. action tubes and higher pipetting errors 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 cone shaped micro well plates You can also obtain MST tested micro reaction tubes from NanoTemper Technologies e Always spin down the stocks your are using labeled and unlabeled molecules for 5 min with 13000 rpm This will remove big aggregates which is one of the main sources for noise Note If your protein sticks to surfaces you may use detergent additives like BSA or low binding reaction tubes to www nanotemper de Fluorescence Counts VNC Ve f _Mreocr 2 2 Quality of your labeling procedure BLUE LED Power 50 N oO oO oO ab gi O oO oO O O oO O oO 50 100 150 Concentration NT495 BLUE nM Figure 2 Example of a calibration curve using NT495 dye with 50 LED power 20 mn 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 preparations of the labeled molecule you can use NanolTemper 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 a prot
8. capillaries you can also try different buffers e g containing detergent BSA casein or other additives Additionally try to improve buffer conditions by adjusting pH and ionic strength www nanotemper de VNC Va l eMP erre 3 3 Sample Quality How can I find the Best Buffer Composition Fluorescence O o oo Cycles Evaluation Points a Hot Cold 10 10 10 Concentration Figure 5 1 The graph shows 4 times the same sample measured with 40 MST power The sample quality is very poor The inhomogenity of sample is clearly seen by the bumpyness of the MST curves aggregation The Thermophoresis with Jump result shows a noise of 10 units Sample quality needs to be improved before performing MST experiments Fluorescence o o Cycles Evaluation Points 767 0 766 5 Hot Cold 766 0 765 5 765 0 40 10 10 Concentration Figure 5 2 The graph shows 4 times the same sample measured with 40 MST power The sample quality is very good There are no bumps in the curves The time traces almost perfectly overlap The Thermophoresis with Jump result shows a noise of less tan 2 units You are ready to start an MST experiment Note The sample shown in Figure 5 1 and 5 2 is the same In Figure 5 2 measurement was performed in MST optimized buffer and after a centrifugation step 13 000 rpm for 5 min Up to now you have c
9. d quality of your sample 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 Also a centrifugation step 13 000 rom for 5 min in order to remove aggregates helps to improve sample quality Note Standard buffer recommendation MST optimized buffer 50 mM Tris pH 7 4 150 mM NaCl 10 mM MgClz 0 05 Tween 20 If no improvement could be observed using this buffer please test different buffers as HEPES Tris or phosphate buffers You can 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 thermophoretic amplitudes should be tested in enhanced gradient capillaries as well 10 www nanotemper de 4 Assay Setup A Prepare a serial titration of the unlabeled molecule Highest concentration unlabeled molecule 20 pl Add 10 pl buffer to 15 reaction tubes Transfer 10 pl from wal 1 to wal 2 mix well Transfer 10 pl from wal 2 to wal 3 mix well Repeat this steps in all wals ry Load samples and start MST analysis Figure 6 Schematic overview how to prepare a MST experiment Now you are ready to start your MST experiment You have a homogenous stable sample that has a low baseline noise This allows you to detect even minute changes in the thermopho
10. e bound or unbound state is lost due to unspecific adsorption precipitation during sample preparation or when filling the capillary This leads to a concentration dependent change in fluorescence within the serial dilution This effect can generate false positive thermophoretic signals In order to avoid unspecific adsorption precipitation supplement the assay buffer with passivating agents like BSA 0 05 mg ml detergent 0 05 Tween 20 or test different buffer compositions by varying pH or ionic strength To rule out sticking and to prove a binding event a mandatory capillary scan gt 15 min after filling the capillaries has to be performed for the capillaries with low fluorescence intensities max 2 capillaries to obtain a good resolution of peak shape If sticking peaks with shoulders or U shape is observed the assay has to be optimized see 3 3 and data cannot be used In this case improve the assay by working at a constant level of BSA e g 0 1 0 5 mg ml or Tween 20 up to 0 05 Note Additional negative controls comprise a non binding molecule with the molecule of interest or a binding deficient mutant of the molecule of interest 12 www nanotemper de VNC Va f _Mreocr 5 2 Analysis of Thermophoresis Signals Evaluation Points The standard setting for evaluation is the analysis mode Thermophoresis There are different rules to access the quality of your MST data amp 930
11. ein the protein preparation has to be as pure as possible For the same reason avoid carrier proteins as BSA in the protein stock Other proteins that get labeled as well will reduce your signal to noise ratio as the free dye does 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 5 nM 6 25 nM 3 12 nM 1 56 nM Use your interaction buffer to prepare the serial dilution of the dye fill these samples in standard capillaries and start a Capillary Scan with 50 LED power Then check the fluorescence intensity in each capillary 2 Determine the concentration of your labeled molecule Prepare a 100 ul dilution of 50 nM of the labeled molecule in your interaction buffer and fill it into a single capillary Then start a Capillary Scan at the capillary position with 50 LED power Use the fluorescence value from the dye calibration curve step 1 in order to estimate the concentration of your labeled molecule IMPORTANT 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 but a very low labeling efficiency might also indicate a problem with protein activity Too
12. escence counts Note It is important to measure with 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 standard capillary and start the Capillary Scan with 50 LED power 3 Compare the intensity to the dye calibration curve you prepared previously Note As described before the fluorescence intensities should match the expectations from the calibration curve by a factor of 2 3 If this is not the case 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 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 with less than 200 fluorescence counts Never perform MST experiments if the fluorescence intensity is higher than 1500 counts To achieve this the sample concentration can be adjusted accordingly or the LED power should be varied between 15 and 95 Note For high affinity interactions Ka lt 10 nM the concentration of the molecule should be on the order of the Ka or below If the Ka 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 with 95 LED power Once you
13. hosen a suitable concentration of the labeled protein sample and you have tested in which capillary type your sample is stably in solution In this chapter you will learn how to find the most suited buffer for your MST experiment that guarantees good reproducibility of MST results This is the case when all time traces are well overlapping for the same sample The most straight forward test for the buffer quality therefore is to compare the time traces obtained in gt 4 capillaries filled with exactly the same sample To find the best buffer composition please follow the following steps 1 Prepare 100 ul of the sample in your binding buffer and 100 ul of the sample in MST optimized buffer 50 mM Tris HCI pH 7 4 150mM NaCl 10 mM MgCle 0 05 Tween 20 2 Fill the type of capillary you determined in step 3 2 using the sample stocks prepared 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 MST power Load the results in the analysis software and select the Thermophoresis with Jump tab for analysis The data should have a noise of 4 units or less IMPORTANT If the noise is more than 8 units we strongly recommend testing different buffers to improve the results A rule of thumb A decimal on the y axis of the Thermophoresis with Jump plot proves a goo
14. mat or improve the buffer composition You can learn more how to find the best buffer in step 3 3 IMPORTANT Some molecules will stick to the surface of capillaries The resulting MST signal has a poor quality NanoTemper offers different types of covalently polymer coated capillaries to avoid any unspecific sticking to the glass surfaces These are called hydrophilic or hydrophobic capillaries NanoTemper also offers a Capillary Selection Kit which contains all important capillary types For more information visit our homepage www nanotemper de To test the best capillary type please follow the following steps 1 Prepare 120 ul of the labeled molecule at the concentration you want to use in the assay determined in step 2 and 3 2 Fill four standard treated capillaries four hydrophilic capillaries and four hydrophobic capillaries with the sample from step 1 3 Put these 12 capillaries on the tray insert it into the instrument and start a capillary scan using the LED settings determined in step 3 1 Note The capillary scan starts at the back of the tray position 16 or 12 respectively Take this into account when you choose the type of capillary for your experiment The following graphs show examples of stable and sticking samples 4 Ifthe fluorescence peaks of the scan are symmetrical you can use these capillaries and go on to the next step Note In the unlikely case that the sample is sticking to all types of
15. r 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 10 nM 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 8 www nanotemper de VN Ye zMPzZr 3 2 Capillary Check Which MST Capillary Type should I use Fluorescence Figure 4 1 No sticking Symmetrical fluorescence peak You can use this capillary type for experiments Fluorescence Figure 4 2 Slight sticking Shoulders in fluorescence peak Please note that it might take 5 minutes to observe an obvious sticking effect If you are not sure repeat the scan after 5 upt to 15 minutes or after the MST measurement a 8 o 50 Fluorescence N wo te S S 8 8 Figure 4 3 Strong sticking Shoulders in fluorescence peak increase You MUST test another capillary types or improve the buffer composition see step 3 3 Fluorescence Figure 4 4 Very strong sticking A clear double fluorescence peak appears You MUST test another capillary for
16. resis of your molecule of interest upon interaction with its partner of interest 1 Prepare 16 small micro reaction tubes best suited are tubes with a volume of 200 ul or less Label them from 1 through 16 2 Fill at least 20 ul of the highest concentration you intend to use in the first micro reaction tube 1 3 Fill 10 ul of the optimal assay buffer into the micro reaction tubes 2 to 16 Note Avoid any buffer dilution effects The buffer in tube 1 and the buffer in the tubes 2 16 must be the same Otherwise you get a gradient in salt DMSO glycerol or other additives This interferes with the results you will obtain from the MST measurement 4 Transfer 10 ul of tube 1 to tube 2 and mix very well by pipetting up and down several times Note In order to avoid bubbles or droplets etc we recommend to carefully pipett up and down several times and not to vortex these small volumes that may also lead to denatured protein or sample 5 To get a serial dilution repeat step 4 15 times and remove 10 ul from tube number 16 after mixing 6 Mix 10 ul 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 min incubation at room temperature are sufficient 11 www nanotemper de VNC Ve f _Mreocr
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