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Monolith NT.115

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1. 0 7 0 8 0 9 Measurement No 1 Dilution 11 z 4 0 5 3 0 6 Position mm From 1 In the Beginning check the following in the top line of the window Connection At Connection there must appear the green check mark Status Ready At Status there must appear Ready User Manual Monolith NT 115 Date July 2011 Version 03 41 Wwww nanotemper de 49 0 89 4522895 0 VNC TS T MPzer User Manual technologies 7 1 Setting Parameters Fluorescence LED Selection and LED Power gt LED LED Selection Red gt LED Power EEE 20 Optimal excitation of GFP FITC FAM YFP Alexa488 and similar RFP HEX Cy3 and similar red Cy5 Alexa647 Atto647 Dy647 and similar First select the fluorescence excitation wavelength for your fluophore at LED Selection e g Red for NT 647 dye The LED Power can be varied to 100 max 240 mV A good starting value is 50 Vary the LED power until the maximum fluorescence of the capillaries is between 80 and 1500 counts optimal 200 1000 When using a high concentration of labeled sample e g 10 UM you can choose a less optimal filter setting e g blue excitation and red detection for a red dye This allows to measure highly intense sample without saturating the
2. a o c a in 350 lo Oo u a o o h a o Deselect all Extrapolation to gt Hot Cold 10 10 Concentration Now the Calculated Curve shows the remaining measurement results in an optimal scale Please note that you need to remove outliers to obtain a good fit User Manual Monolith NT 115 Date July 2011 Version 03 65 Wwww nanotemper de 49 0 89 4522895 0 VNC Ye TcMPER User Manual technologies 9 4 Save MST Data 9 4 1 Save as an image file Click right mouse button and choose Save Image As to save the image as a picture Evaluation Points Fitted Curves O LED 25 Laser 17 mm Fit Kd Fit Copy Save Image s Page Setup Print Show Point Values Un Zoom Undo All Zoom Pan Set Scale to Default Concentration 9 4 2 Save as a text file To save the data as a text file choose Save Data and select a path The text file can be opened with most common statistics graphics software s as MS Excel Origin Prism and Sigma Plot 9 5 Generation of MST Report It is possible to generate a quick report of the analyzed data which contains the following information experiment name date Laser amp LED Power MST curves MST fitted data as a binding curve Thermophoresis or Temperatur jump or Thermophoresis with Jump or Hot Cold calculated kD and a comment You can easily generat
3. Prepare a serial dilution of the not labeled binding partner and fill 10ul of each sample of the dilution series in to 16 small micro reaction tubes Fill 10 ul of a 2x stock solution of the labeled binding partner into the same 16 tubes and mix very well Incubate the samples in the dark Incubation time and temperature can differ between different molecules and should be tested in a pilot experiment by the researcher In most cases 30 60 minutes incubation at room temperature are sufficient Transfer the binding reaction into Monolith NT Capillaries After an incubation time sufficient for the reaction to reach equilibrium 5 ul of each binding reaction is aspirated into Monolith NT Capillaries see chapter 5 User Manual Monolith NT 115 Date July 2011 Version 03 31 Wwww nanotemper de 49 0 89 4522895 0 VNC aD MPE User Manual Cf hnolo es KD of binding of molecule X to Y 50 nM Titration series range 1000 nM 0 025 nM Dilution Conc Y Concentration of Binder Y 1 mM in Buffer YB series nM Prepare the highest concentration of the dilution series by adding 1ul of Compound Y to 0 5ml buffer of choice 2uM This dilution No 1 still contains traces of buffer YB Prepare the Series dilution buffer SD buffer that has the same constituents the dilution No 1 has e g DMSO Glycerol etc Transfer 15ul SD buffer to 15 0 2ml tubes No 2 16 Transfer 15ul with a clean tip from the first 0 2ml vi
4. VN Ye TEMPER User Manual technologies Microscale Thermophoresis can be used to NanoTemper s unique MST technology allows to measure affinities KD dissociation constant between any bio molecules directly in bioliquids study the effect of serum cell lysate or other bioliquids on biomolecules and to separate multimerization aggregation and other artifacts from true binding events study membrane bound proteins directly in membranes study multi component reactions or biochemical fractions containing enzymatic activities complex formation order of assembly interfering factors in the medium access larger screening projects in a label free manner using fluorescently labeled competitors Competitive MST discriminate between different binding sites on a target of interest study the enzyme kinetics vmax kcat study the stoichometry and determine the number of binding sites of biomolecules study the binding ernergetics AG free energy AH enthalpy and AS entropy study the inhibitor affinity Ki either directly or in a competition experiment No Binding Binding IR Laser Focus IR Laser Focus Fluorescence Signal Figure 2 Scetch of the thermophoretic effect Binding of an antigen to a fluorescently labeled antibody changes its thermophoretic motion in the temperature gradient For example the complex moves from lower to higher temperatures while the free
5. is bumpy This is because aggregates are transported into the measurement volume by a convective flow induced by local heating In such cases Centrifuge you sample before using it in a MST experiment 5 minutes 13 000 rpm 4 C Actually we recommend to centrifuge any stock of the sample before it is used in MST You may also use detergents such as Tween 20 0 01 0 1 or Triton X 100 0 01 0 1 User Manual Monolith NT 115 Date July 2011 Version 03 26 Wwww nanotemper de 49 0 89 4522895 0 OONO MPzer User Manual technologies No Aggregates 1560 1520 1480 1440 1400 1360 Fluorescence 1320 1280 1240 4200 2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 4 Time seconds Some Aggregates better sample preparation needed 1100 1050 qu oO o O 1000 u 950 900 2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 4 Time seconds Many Large Aggregates better sample preparation needed Ju 370 360 350 340 330 Fluorescence 320 310 300 2 0 2 4 6 8 10 12 14 16 18 20 22 24 2 28 30 32 34 36 38 40 4 User Manual Monol Time seconds www nanotemper de 49 0 89 4522895 0 vo TcMPER User Manual technologies 3 4 Quick Start Protocol for Sample Preparation Prerequisite and general remarks One substrate should be fluorescently labeled Make sure that there isn t any not react
6. Capillaries 50 Monolith NT Standard Treated Starter Set Capillaries 50 Monolith NT Hydrophobic Capillaries 50 Monolith NT Hydrophilic Capillaries Sealing Wax Treated Capillaries Sealing Wax K003 Monolith NT Hydrophobic 200 Capillaries K004 Monolith NT Hydrophilic 200 Capillaries User Manual Monolith NT 115 Date July 2011 Version 03 25 Wwww nanotemper de 49 0 89 4522895 0 OONO T MPEs User Manual technologies NOTE Random variations of fluorescence counts can also be caused by adsorption of the biomolecule to the micro reaction tubes In this case a detergent or BSA may be used a higher sample volume may be prepared or low adsorption micro reaction tubes may be deployed 3 3 Checking the Sample Quality Detecting Aggregation In addition the potential aggregation behavior of the biomolecule of interest can be tested with microscale thermophoresis Often protein preparation contain aggregates that are very difficult to observe with standard methods To test for the quality of your preparation perform the following experiment with your molecule of interest at working concentration and in the intended buffer Start a measurement with the following setting LED power adjusted to you sample concentration Laser power 40 Laser on time 30 seconds Laser off time 5 seconds In case that the biomolecule of choice aggregates the fluorescence signal during the laser on time
7. Please rule out that this is not due to a shift in buffer composition by preparing your sample carefully After finding the capillaries it s necessary to check the numbers of capillaries and to apply with the OK button CapSearchType 17 816 13 818 119 820 25 857 31 878 137 876 ss 49 878 55 881 61 914 167 917 73 859 Capillary Search Type by maximum values co a tn dm t to toi Threshold 50 pi pami eet NO t a i There are two algorithms a By maximum values standard b By threshold this is recommended when there is Low fluorescence intensity Or more than 50 difference in fluorescence intensity between different capillaries User Manual Monolith NT 115 Date July 2011 Version 03 49 Wwww nanotemper de 49 0 89 4522895 0 VNC Ve TEMPER User Manual technologies Data Saving Use the New Project button in order to generate new Project files You can choose the Load Project button in order to attach a new measurement to an existing Project file In the Experiment s Name field you can type an experiment name Multiple experiments can be saved in the same Project file Project NewProject LoadProject Experiment s Name The data analysis software recognizes then automatically these results in the file and plots the MST results vs Concentration Th
8. if 0 0 rasta ef teh Ka leg tcf Ka ached Ca i The value is kept constant and is known as well as the value which is changed during the titration series The fraction of bound molecules x can be dervied from F en ge PR a Foul Where Frorm A is the normalized fluorescence of only unbound labelled molecules A and Frorm AT is the normalized fluorescence of complexes AT of labeled User Manual Monolith NT 115 Date July 2011 Version 03 54 www nanotemper de 49 0 89 4522895 0 VNC Ye TcMPER User Manual technologies molecules bound to their targets in saturation every labeled molecule bound to a target is the measured value for the respective concentration inthe titration series For the value equals and in the case of all labeled molecules A bound to their targets T x 1 equals In the cases where there is more than one binding site and the binding curve cannot be fitted with the formula for Kg the Hill equation can be used 1 TR ir er Y where n is the hill constant describing the cooperativity of the binding and the ECso is the concentration at which half of the molecules are bound PLEASE NOTE In the Analysis Software the value is plotted as the abscissa User Manual Monolith NT 115 Date July 2011 Version 03 55 www nanotemper de 49 0 89 4522895 0 ONNO T MPzer technologies User Manual 9 Analysis Software The analysis software allows a quick and a precise
9. 2 2 Application Range Monolith NT 115 allows to measure the binding of all kinds of biomolecules KD 1 nM 500 mM as well as the activity of enzymes Higher Affinities are accessible in competition experiments 2 3 Sensitivity Monolith NT 115 can measure as little as the binding of single ions 40Da or small molecules 300Da to a target as well as the binding of large complexes such as ribosomes 2 5MDa 2 4 Sample Consumption The Monolith NT 115 requires only little sample material gt 1 nM of the labeled compound and a titration of the unlabelled compound in the range of factor 10 20 of the expected dissociation constant For standard applications 5ul of sample material is filled in the capillary 2 5 Capillary Format The capillary format used for the Monolith NT 115 is inexpensive easy to handle and offers a maximum of flexibility in the experiment scale The standard sample tray format allows to process automatically up to 16 capillaries e g for a detailed KD analysis or alternatively to perform smaller pilot experiments involving only 2 3 sample concentrations User Manual Monolith NT 115 Date July 2011 Version 03 22 Wwww nanotemper de 49 0 89 4522895 0 VNC Ve T MPES User Manual technologies 2 6 Close to Native Conditions Monolith NT 115 can monitor the binding and the biochemical activity of biomolecules under close to native conditions immobilization free Ina solution of choice rang
10. This symbol indicates laser radiation it is put on products which have a laser built in This warning label is positioned at the backside of the device er INS 4 Identification label This label is positioned at the backside rear panel Manufactured by Voltage 12V DC NanoTemper Technologies GmbH Polarity amp Floessergasse 4 max Current Input 5 0 A of the device 81369 Muenchen Germany www nanotemper de Made in Germany SN 201107 BG 001 c User Manual Monolith NT 115 Date July 2011 Version 10 6 www nanotemper de 49 0 89 4522895 0 TVN T MPE E User Manual tschnalooies WARNING Do only operate the Monolith NT 115 instrument with the delivered external power supply Power Solve PSG60 12 02 or Sinpro Electronics Power Supply SPU63 105 Do only use the delivered cables and plugs If not doing so you risk electric shock and fire WARNING Do not operate the Monolith NT 115 with substances and under conditions which do cause a risk of explosion implosion or release of gases Only operate the Monolith NT 115 with aqueous solutions CAUTION Ensure that the power plug of the external power supply is well accessible The Monolith NT 115 instrument has to be installed in a way that it does not hinder the access to the external power supply and its power plug CAUTION The weight of the Monolith NT 115 instrument is approx 22 kg do not move the instrument alone two persons required for transpo
11. VN Ve TEMPER User Manual C AN Li QIES u A Powerful Technology for I T Be the Analysis of Biomolecules Monolith NT 115 User Manual User Manual Monolith NT 115 Date July 2011 Version 10 1 www nanotemper de 49 0 89 4522895 0 VNC Ye TEMPER User Manual technologies Contact NanoTemper Technologies GmbH www nanotemper de Floessergasse 4 D 81369 M nchen info nanotemper de phone 49 89 4522895 0 fax 49 89 4522895 60 User Manual Monolith NT 115 Date July 2011 Version 10 2 www nanotemper de 49 0 89 4522895 0 VN ve TEMPER User Manual technologies Table of Contents SAleLy GO MSC CL ADIDAS a E sana auuesauentqunenensunacneds 6 Regulatory State MENi Naren a mm mm mT ORE ES E eee earn eee 9 SOC IC AON Se ee re da Ent 10 DEC UC ri id gneeadrentnrnead E AE EE 12 OCO atra ee crac emt een ea oe act eee 12 PPV V2 5 116 y AN a A A E A 13 instalation Regui remeis ee 14 Installation and Connecting Cables sisses i e a aE AE E 15 Maintenance and Operan Seesen T E ETE T E ET T 16 MV ASUS LTE IL CIN e E E E E E 17 1 Introduction to Microscale Thermophoresis MST cccccccssssccccecssseccceeeesecceeeeeesecceeseeeeeceseeeeeeeeeees 18 2 M nolth NT 115 Instrume ee ee ee 22 21 MO ee nen oracao 22 2 2 ADDNCILON R ANS ea ee 22 ZI SINS IV ee ee ee een 22 2A Sample Consumo ee ee ee ee ee 22 2 5 CAD WARY FORAL rer ee 22 2 6 Close to Native Conditions ccccecccccccccccc
12. a good choice 7 Select the Set Temperature and press Switch on to start fitting the temperature For the most measurement the room temperature is ok so there is no adjustment needed at this part 8 Select the IR Laser power values between 10 and 100 are possible good choices are 15 40 and 100 We strongly recommend to use all three laser powers when analyzing a unknown system 9 Type in the concentration for each capillary Capillary No 1 should contain the highest concentration No 1 is the first capillary from the operator s point of view The Name column accepts additional information like the order of magnitude of concentration e g nM 10 Select the directory and file name to save the data 11 Press Start to start the measurement a window will appear asking for the measurement replication A value of 2 means each capillary will be measured after completion of the first round of measurement The delay defines the time between measurements User Manual Monolith NT 115 Date July 2011 Version 03 52 Wwww nanotemper de 49 0 89 4522895 0 vo TcMPER User Manual technologies 8 Data Analysis Theoretical Background The Microscale Thermophoresis of the fluorescently labeled molecules is measured by monitoring the fluorescence distribution F inside a capillary A microscopic temperature gradient is generated by an IR Laser which is focused into the capillary and is stron
13. and Connecting Cables Preparation Prepare a table which can bear a weight of about 50 kg and has a free area of 80 cm width x 50 cm depth Put the Monolith NT 115 instrument and the Notebook to this free area CAUTION The weight of the Monolith NT 115 instrument is approx 22 kg do not move the instrument alone two persons required for transport movement If you move the instrument alone it poses a risk of personal injury or damage to the instrument Connecting the Power Supply and the Monolith NT 115 Confirm that the power switch of the Monolith NT 115 instrument is off power switch is at the backside left of the instrument WARNING Do only operate the Monolith NT 115 instrument with the delivered external power supply Power Solve PSG60 12 02 or G nter Power Supplies SPU63 105 Do only use the delivered cables and plugs If not doing so you risk electric shock and fire Connect the external power supply to the electrical socket Then connect the power supply to the Monolith NT 115 instrument CAUTION Ensure that the power plug of the external power supply is well accessible The Monolith NT 115 instrument has to be installed in a way that it does not hinder the access to the external power supply and its power plug Connect the Monolith NT 115 instrument to the Notebook by using the delivered network cable Switch on the Monolith NT 115 and the Notebook User Manual Monolith NT 115 Date July 2011 Version 10 15
14. binding partner Ideally the ratio of label to protein DEG degree of labeling is 1 1 or less Unbound label has to be eliminated from the labeling mixture completely The concentration of the protein should be close to the expected KD or less and should yield a range of 80 1500 fluorescence counts typically equivalent to 1 200 nM when measured with the Monolith NT 115 The following kits offer optimized fluorescence labeling and purification protocols for use in Microscale Thermophoresis The dyes are widely tested with thermophoresis and the protocols are optimized to remove free unreacted dye User Manual Monolith NT 115 Date July 2011 Version 03 30 Wwww nanotemper de 49 0 89 4522895 0 VN ve TEMPEAR User Manual technologies LOO1 Monolith NT Protein Labeling Kit RED 4x 100 500 ug Protein LOO2 Monolith NT Protein Labeling Kit GREEN 4x 100 500 ug Protein L003 Monolith NT Protein Labeling Kit BLUE 4x 100 500 ug Protein Prepare the unlabeled binding partner Prepare a 16step dilution series around the expected KD starting from a concentration of 20fold the expected KD down to 0 02 fold the expected KD Prepare the samples with a twofold higher concentration since mixing volume ratio of 1 1 decreases the concentration by a factor of 2 Use sufficiently small micro reaction tubes to avoid evaporation of the sample e g with a volume lt 100ul when preparing 10 20ul of sample Binding reaction
15. detector This setting can also be used for FRET experiments NOTE The maximum value for the fluorescence is 2500 this is the saturation value of the detector The LED power has to be chosen in such a way that the fluorescence is below 2500 otherwise the MST Signal of the molecules cannot be measured For optimal results chose fluorescence intensity between 80 1500 counts optimal 200 1000 counts User Manual Monolith NT 115 Date July 2011 Version 03 42 Wwww nanotemper de 49 0 89 4522895 0 VNC Ye zmPzEr User Manual technologies Overexposure too much LED Power 2400 2100 1800 Fluorescence ee 2S So fes So o Co o 72 6 72 9 73 2 73 5 73 8 74 1 74 4 74 7 75 75 3 Position mm Underexposure too low LED Power Fluorescence a N N FS amp gt gt 8 mn N e co N a gt oo N 72 6 72 9 73 2 73 5 73 8 74 1 74 4 74 7 75 75 3 Position mm N Good LED Power 80 1500 counts 1000 Fluorescence N A on qo o S S 8 3 w oO 29 2 29 6 30 30 4 31 32 32 4 32 8 30 8 1 2 Position mm User Manual Monolith NT 115 Date July 2011 Version 03 43 Wwww nanotemper de 49 0 89 4522895 0 VN Ve TEcMPER User Manual technologies Thermophoresis IR Laser Power and IR Laser On Time LASER Laser On Time 30 s Laser Power Final Laser Off Time 5 s 4 10 With the Laser On Time the time during which the MST Signal is observed can be defi
16. evaluation of Thermophoresis data and quantification of kD After starting the program following screen window appear a We TzMPZrTr technologies Update Mode C Users nanotemper Desktop Measurement Nr Guri 7 Distinguish Runs la asian a Cursor Hono Hot Cold Thermophoresis Thermophoresis with Jump Temperature Jump Fluorescence Evaluation Points Thermophoresis with no Jump PSE Show Report 1 Border 1 E A Kd Fit Hill Method Kd Border Hill Border 10 Concentration Ka Concentr Unbound Bound Fix E Fix F Fix E Fix User Manual Monolith NT 115 Date July 2011 Version 03 56 Wwww nanotemper de 49 0 89 4522895 0 VNC Ye TEMPER User Manual technologies 9 1 Update and Load function 9 1 1 loading data in Real Time During a measurement you can the evaluate results of the already measured capillaries To do this you should activate the update mode by pushing the Update Mode button Load the actual ntp file the file with the largest number in the corresponding directory Now you have only to push Update and the software is loading all new values for you C Update Mode Update 9 1 2 Load Measurement When pushing the Load button measurement data can be imported You must deactivate the Update Mode before VNC ve l eMP erR technologies The file ntp for t
17. finder tool ussssnsnssnssessssssnnneneennnennnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn essen 45 73 CONCENU AVON NOU nina aaa 47 74 Instrument Control FUNCTIONS nun 48 7 5 Quick Protocol Microscale Thermophoresis Measurement ccccccceeccceeeceeeecseeeseeeeeseeneeeees 52 3 Data Analysis Theoretical Background een 53 9 Allalysis SOHN WATe nee enge 56 e IO ole abe and Load TUNIC UO coc ee ee ee 57 9 1 1 loading data In Real TIME u uuenun een 57 9 1 2 Load M essurement nee ee eisen 57 9 2 MSE Ost a analy SIS anne een een 59 9 2 1 Temperature jump and Thermophoresis Standard ccccccscsseecscesecseeeeseseeeeeseeeeeeseeees 60 9 2 2 Only Thermophoresis a ee eins 60 9 2 OV Temperat Urea ee er 61 9 2 4 Hot Cold Deactivation of the automatically analysis serena 61 2 5 Cola FUO ESCE Ne ee ee ee ee eine er 62 IL UNS FRUN nen een re TE Re Le TER nee 62 9 3 Data DON TEO a ee ge 64 TANEN TO aaa asio sro sang oc good eaten OBEREN ERBE U Gado Rods coa il a ndo ss NSEREOEREDERN HEIEHERTDERTEELERECEEECUREERERGSE 66 User Manual Monolith NT 115 Date July 2011 Version 10 4 www nanotemper de 49 0 89 4522895 0 VN Ye T MPzEr User Manual technologies AT VEe AS ANIMALE TIS aque cesso nai SEER OUSOU ao 66 9 2 25 390 as ae ancas sa Ai ie eee eC eee et dar SD a eee 66 9 5 Generation o MST Report unos sab ee SS SS ee ae 66 10 Data Processing of Multiple Experiments cccccssscccccsseccceesscce
18. instrument Do not try to repair the instrument by yourself Do contact the NanoTemper service for repairing the instrument CAUTION The manual opening of the instrument is not allowed Manual opening pose a risk of personal injury or damage to the instrument Contact NanoTemper service personnel if you need to open the instrument User Manual Monolith NT 115 Date July 2011 Version 10 16 www nanotemper de 49 0 89 4522895 0 VNC Ye TEMPER User Manual technologies Waste Treatment Waste treatment is your own responsibility You must hand it to a company specialized in waste recovery Do not dispose the instrument in a litter bin or at a public waste disposable site For detailed information please contact the NanoTemper service User Manual Monolith NT 115 Date July 2011 Version 10 17 www nanotemper de 49 0 89 4522895 0 VNC TS TEMPER User Manual technologies 1 Introduction to Microscale Thermophoresis MST Microscale Thermophoresis is a powerful new technology and easy to handle It measures changes in the hydration shell of molecules and allows to quantify enzyme kinetics or to measure biomolecule interactions under close to native conditions Virtually any biochemical process relating to the interaction size Stability or conformation of biomolecules and biochemical complexes can be measured with high sensitivity 150 years ago Charles Soret and Carl Ludwig 1 discovered that molecules move in
19. modifying a DNA molecule also an intercalating dye can be used to monitor the thermophoretic changes When preparing an experiment to analyze the enzymatic activity the ratio between substrate and enzyme has to be carefully chosen Too high enzyme concentration or low amount of substrate will lead to a very fast conversion of the substrate to the reaction product It is recommended to slow down the reaction kinetics to gt 10 minutes in order to be able to start the analysis before a significant part of the substrate is already converted Since enzyme kinetics vary between different enzyme reactions it is recommended to adjust the reaction conditions and the concentration of enzyme and substrate In order to reach optimal results use a low amount of enzyme and an excess amount of substrate It is recommended to start a new experiment with 1 10 nM enzyme or less and 10 uM substrate NOTE Use only a little amount of enzyme and use substrate in excess You can mix fluorescently labeled substrate and not labeled substrate if the conversion of these molecules to a product is similar To analyze an enzymatic reaction you should at least prepare two samples One containing the substrate and all cofactors necessary The second negative Control should contain the substrate but it should lack an essential cofactor e g ATP S adenosylmethionin etc or contain an inhibitor Prepare approx 10 ul of these samples Add the enzyme to both soluti
20. of opposite sign In the following a few examples are shown all of them lead to very good results when analyzing the MST Signal Positive T Jump and positive Thermophoresis 5 E i User Manual Monolith NT 115 Date July 2011 Version 03 69 Wwww nanotemper de 49 0 89 4522895 0 VNC Ye TcMPER User Manual technologies Negative positive T Jump and negative positive Thermophoresis normall d Ruore menos R B 5 E 8 Note If your signal difference between bound and unbound state is less than 10 units you will not see a separation between individual curves by just looking at the traces However to judge the quality of an experiment the amplitude is not the main criterion More important is the ratio between signal to noise that means the noise in the baseline of the signal compared to the difference between bound and not bound state See also in the MST Protocols document User Manual Monolith NT 115 Date July 2011 Version 03 70 Wwww nanotemper de 49 0 89 4522895 0 VNC TS T MPzr User Manual technologies 12 Application Protocols SAMPLE PREPARATION PROTOCOL of Monolith NT Control Kit RED Cat Nr C030 1 Make 1 x reaction buffer by diluting the supplied 5 x reaction buffer with water Add 280ul ddH20 to 5 x buffer 2 Thaw vial B AMP Stock and spin down all liquid to the bottom of the vial Make a serial dilution 12 AMP dilutions Add 30ul of AMP
21. or particles are initially distributed evenly and diffuse freely in solution By switching on the IR Laser the molecules experience a thermophoretic force in the temperature gradient and typically move out of the heated spot In the steady state this molecule flow is counterbalanced by ordinary mass diffusion After turning off the laser the particles diffuse back towards a homogeneous distribution Different kinds of information can be observed from such experiment Fluorescence signal before turning the laser on fast temperature dependent changes MST T Jump in fluorescence intensity of the analyzed molecules thermophoresis and back diffusion after switching the laser off The Microscale Thermophoresis Technology can detect even weak binding events which are typically difficult to access like the binding of small molecules or single ions to proteins 3 4 Furthermore it can be used to study the activity of molecules including enzymatic modifications of substrates or competitive binding of inhibitors and substrates to an enzyme The interaction of biomolecules small molecules DNA RNA proteins peptides sugars lipids ribosomes etc can be measured under close to native conditions immobilization free ina solution of choice ranging from standard and proprietary buffers to complex bioliquids including blood serum or cell lysates User Manual Monolith NT 115 Date July 2011 Version 03 19 Wwww nanotemper de 49 0 89 4522895 0
22. temperature gradients a physical effect called thermophoresis Extensive research conducted at the Biophysics Department of the Ludwig Maximilians University Munich LMU identified the solvation entropy and the hydration shell of molecules as the driving force 2 Today with the availability of IR lasers precise temperature gradients on the microscale can be rapidly induced within thin glass capillaries enabling the study of biomolecule interaction and activity User Manual Monolith NT 115 Date July 2011 Version 03 18 Wwww nanotemper de 49 0 89 4522895 0 OONO T MPE User Manual technologies 96 EEE Initial Fluorescence B Initial no molecule flow ack State MST T Jump diffusion no molecule flow 90 9 es Fluorescence Detection j IR Laser IR Mirror 4 gt Thermophoresis Backdiffusion o molecule flow Molecules 1 04 nn p Optics Thermo phoresis Capillary 0 9 Normalized Fluorescence 0 8 10 20 30 40 50 IR Laser on Time s IR Laser off Figure 1 Microscale Thermophoresis A fluorescence excitation light source and detector is used with an additional hot mirror inserted into the path of fluorescence light With this mirror the heating IR Laser couples into the path of fluorescence light and is focused with the same objective used for fluorescence detection This allows observation of thermophoresis in thin glass capillaries Fluorescently labeled molecules
23. the x axis displays the time in seconds The overall time consists of 5 seconds in the beginning cold fluorescence followed by Laser On Time and Laser Off Time 1880 1840 1800 1720 1680 1640 1600 1560 2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 4 Time seconds o Fluorescence User Manual Monolith NT 115 Date July 2011 Version 03 51 www nanotemper de 49 0 89 4522895 0 VNC eD TcMPER User Manual technologies 7 5 Quick Protocol Microscale Thermophoresis Measurement 1 Atthe very beginning press Open Close on the Touchscreen and put in the tray with your samples 2 Select the appropriate excitation LED for your fluorescence dye 3 Select the excitation power of the LED 50 will be a good choice to start 4 Select the start and end position for the capillary scan to find the capillaries then press Find Capillaries The instrument will then scan for the capillaries and will show them on the Fluorescence display If the maximum fluorescence of the capillaries is between 200 and 1500 the LED Power setting is ok If the fluorescence is 2500 your excitation power or the concentration of labeled molecules are too high In this case lower the concentration or press Stop adjust the LED Power and press Find Capillaries again 5 Select the IR Laser On Time 30s is a good choice 6 Select the IR Laser Off Time 5s is
24. time when you have set the temperature controller to high temperatures CAUTION Only NanoTemper stuff is allowed for servicing and opening of the instrument Turn off the power switch and unplug the power cord before servicing the instrument unless otherwise noted Connect the equipment only to the delivered power source Do not use extension cords Have an electrician immediately replace any damaged cords plugs or cables Not doing so poses a risk of personal injury or damage to the instrument User Manual Monolith NT 115 Date July 2011 Version 10 7 www nanotemper de 49 0 89 4522895 0 VNC Ye TcMPER User Manual technologies CAUTION Only open the sample loading site when moving parts in instrument are at rest Do not insert or remove a sample while linear actuator laser or LED is at work Moving parts in the instrument can be harmful CAUTION Do not use the instrument in the cold room CAUTION Turn off the main circuit breaker in the back of the chassis when the instrument is not in use CAUTION Do not use ethanol or other types of organic solvents to clean the instrument as they may remove the instrument paint CAUTION Use the instrument only for biomolecule analytics with aqueous solutions and do not open the instrument on other site than for sample loading CAUTION Only use aqueous sample for analysis in the instrument CAUTION Do not use the instrument with hazardous substances or substances materials whic
25. www nanotemper de 49 0 89 4522895 0 vo TEMP ESsS User Manual technologies Maintenance and Operation Pay attention to the instrument operating environment and always keep it clean so that the instrument can be used in a stabilized condition over a long period Do not place anything on the instrument Cleaning the Monolith NT 115 instrument Switch off the instrument and remove the power plug of the external power supply from the electrical socket Only use dry cloth or cloth wetted with water for cleaning of the instrument CAUTION Do not use ethanol or other types of organic solvents to clean the instrument as they may remove the instrument paint Transporting the Monolith NT 115 instrument Switch off the instrument and remove the power plug of the external power supply from the electrical socket Do not carry the instrument alone two people are need for transportation CAUTION The weight of the Monolith NT 115 instrument is approx 22 kg do not move the instrument alone two persons required for transport movement If you move the instrument alone it poses a risk of personal injury or damage to the instrument Functional Disorder In case of a functional disorder switch off the Monolith NT 115 instrument and wait for five minutes then switch on the instrument again If there is still a functional disorder switch off the device unplug the power cable and contact the NanoTemper service Repairing the Monolith NT 115
26. July 2011 Version 10 11 www nanotemper de 49 0 89 4522895 0 vo Tz MPzr User Manual technologies Preface This manual is your guide for using the Monolith NT 115 and doing Microscale Thermophoresis MST measurements It instructs first time users on how to use the instrument and serves as a reference for experienced users Before using the Monolith NT 115 instrument please read this instruction manual carefully and make sure that the contents are fully understood This manual should be easily accessible to the operator at all times during instrument operation When not using the instrument keep this manual in a safe place If this manual becomes lost order a replacement from NanoTemper Technologies GmbH Notices 1 NanoTemper shall not be held liable either directly or indirectly for any consequential damage incurred as a result of product use 2 Prohibitions on the use of NanoTemper software e Copying software for other than backup e Transfer or licensing of the right to use software to a third party e Disclosure of confidential information regarding software e Modification of software e Use of software on multiple workstations network terminals or by other methods 3 The contents of this manual are subject to change without notice for product improvement This manual is considered complete and accurate at publication 5 This manual does not guarantee the validity of any patent rights or other rights If a Na
27. Monolith NT Capillary In order to avoid experimental artifacts which can be caused by protein adsorbance to the glass capillary or by protein aggregations it is recommended to determine the best Monolith NT Capillary type for a bio molecule of interest by performing the following experiment Load 5 Monolith NT Standard Treated Capillaries with 100 nM or at working concentration of the labeled biomolecule of interest after diluting it with the buffer that is intended to be used in the experiment Place the capillary on the tray and insert the tray in the instrument Chose a folder where to save the scan data and a LED color power User Manual Monolith NT 115 Date July 2011 Version 03 24 Wwww nanotemper de 49 0 89 4522895 0 VNC Ye T MPezr User Manual technologies Close the chassis and start a capillary scan by pushing the find capillaries button If a u shape i e split peak is observed instead of a distinct fluorescence peak it your sample absorbs to the capillary wall See figure below left 3 capillaries Sticking Sample False Capillary Not Sticking Sample ok 1000 800 Q 600 u 2 5 400 LL 200 0 2 4 6 5 10 12 14 16 18 20 22 24 26 28 30 32 Position mm In this case it is recommended to use Monolith NT Hydrophobic and Hydrophilic Capillaries which in almost all cases prevent the absorbance to the capillary glass walls K001 Monolith NT
28. Stock to the first 0 2ml tube Add 15ul selection buffer to the other 11 0 2ml tubes Transfer 15ul with a clean tip from the first 0 2ml tube to the second tube and mix well by pipetting up and down With a clean tip transfer 15ul from the second tube to the third tube and mix well Continue this 1 fold serial dilution until tube number 12 3 Add 105ul selection buffer to vial A Aptamer Stock and spin down all liquid to the bottom of the vial Mix well by pipetting Transfer 10ul of diluted Aptamer solution to 12 clean 0 2ml tubes Transfer 10ul from the AMP serial dilution to each tube in the same order and mix well 4 Load the samples in 12 different NT capillaries and transfer the capillaries to the tray 5 Putthetray in the instrument and find perform the thermophoresis mesaurments Recommended instrumental setting are 20 LED power and 17 IR laser power First time users can find more detailed information how to perform the measurement in the instrument user manual User Manual Monolith NT 115 Date July 2011 Version 03 71 Wwww nanotemper de 49 0 89 4522895 0 VN Ye TEMPER User Manual technologies Data Evaluation Expacted results The binding between the Cy5 labelded DNA Aptamer and AMP show EC50 value of 87 uM Dilutions Concentration AMP 1 5000 uM 2500 uM 1250 uM 625 uM 312 uM 156 uM 78 uM 39 uM 20 uM 10 uM O CON DW UI BW N pa O 1 ATP Aptamer m AMP Sel Buffer O Eos aa O E
29. a O O LL 0 c nM User Manual Monolith NT 115 Date July 2011 Version 03 72 Wwww nanotemper de 49 0 89 4522895 0 VN Ye TEMPES User Manual technologies User Manual Monolith NT 115 Date July 2011 Version 03 73 www nanotemper de 49 0 89 4522895 0
30. al 2uM compound Y from step to the second vial and mix well by pipetting With a clean tip transfer 15ul from the second vial to the third vial and mix well Continue this 2 fold serial dilution until tube number 16 Transfer 10ul from No 1 16 in new reaction tubes and add 10ul of your fluorescently labeled sample e g 100nM to the tube VII Mix very well by pipetting to obtain good results NOTE 1 centrifuge stock solutions before usage to remove aggregates 2 When the highest concentration of binder has a significant concentration of an additional buffer component e g 3 DMSO then the buffer used for dilution has to contain the additive in same concentration The same holds true for differences in ionic strength or pH BSA or glycerol 3 The Monolith NT 115 has a highly precise fluorescence detection and insufficiently mixed samples will be highlighted by random fluctuating fluorescence intensity signals If sample are well mixed a random fluctuation of 10 or even less is achieved User Manual Monolith NT 115 Date July 2011 Version 03 32 Wwww nanotemper de 49 0 89 4522895 0 VNC TS T MPzr User Manual ee 4 2 Preparing an Enzyme Reaction Experiment When analyzing an enzymatic reaction with Microscale Thermophoresis the substrate should be fluorescently labeled e g peptide or DNA A fluorescently labeled peptide can be purchased from various providers In case of an enzymatic reaction
31. ambient humidity exceeds 80 RH condensation may deteriorate optical components 9 Operate the instrument in an atmospheric pressure range of 800 1060 hPa 10 Do not operate the Monolith NT 115 under conditions which pose a risk of explosion implosion or the risk of the release of gases 11 Avoid strong magnetic fields and sources of high frequency The instrument may not function properly when near a strong magnetic field or high frequency source 12 Avoid vibrations from vacuum pumps centrifuges electric motors processing equipment and machine tools 13 Avoid dust and corrosive gas Do not install the instrument where it may by exposed to dust especially in locations exposed to outside air or ventilation outlets 14 For cleaning the instrument only use water 15 Do not install the instrument in a location where it may be exposed to direct sunlight 16 Install the instrument in a horizontal and stable position This includes a table bench or desk upon which the instrument is installed 17 Ensure that no air conditioner blows air directly onto the instrument This may prevent stable measurements 18 Install the instrument in a location that allows easy access for maintenance NOTE The above conditions do not guarantee optimal performance of this instrument User Manual Monolith NT 115 Date July 2011 Version 10 14 www nanotemper de 49 0 89 4522895 0 TEMP ES User Manual technologies Installation
32. antibody moves in the opposite direction Thus the fluorescence increases where the solution is heated when there is a binding event Also a slightly slower or faster motion without change of direction is detectable with thermophoresis User Manual Monolith NT 115 Date July 2011 Version 03 20 Wwww nanotemper de 49 0 89 4522895 0 YN VED T MPzEr User Manual technologies References 1 Ludwig C Sitzungsber Akad Wiss Wien Math Naturwiss 20 539 1856 2 Stefan Duhr and Dieter Braun Why molecules move along a temperature gradient PNAS 103 19678 19682 2006 3 Philipp Baaske Christoph J Wienken Philipp Reineck Stefan Duhr and Dieter Braun Optical Thermophoresis for Quantifying the Buffer Dependence of Aptamer Binding Angewandte Chemie International Edition 49 2238 2241 2010 4 Christoph J Wienken Philipp Baaske Ulrich Rothbauer Dieter Braun and Stefan Duhr Protein Binding Assays in Biological Liquids using Microscale Thermophoresis Nature Communications DOI 10 1038 ncomms1093 2010 User Manual Monolith NT 115 Date July 2011 Version 03 21 Wwww nanotemper de 49 0 89 4522895 0 VNC eD TcMPER User Manual technologies 2 Monolith NT 115 Instrument 2 1 Models Monolith LED LED Blue Dyes Green Dyes Red Dyes Instrument 1 2 NT 115 FITC FAM GFP YFP Cy3 REP mCherry Blue Green NT 115 Green Red NT 115 FITC FAM GFP YFP no detection Cy5 Alexa647 Blue Red
33. ature is plotted TEMPER technologies EI Update Mode Update C Users nanotemper Desktop AMP_test amp_test_ntp ore E a 1X 1 1 case E Cursor Memory 9 2 4 Hot Cold Deactivation of the automatically analysis To choose individual values for cold and hot areas choose the tab Hot Cold on the left side Then move the Cold Cursors to a point on the time trace a few seconds after the heating laser was turned on The position of the Hot and the cold cursors is shown in the graph By pushing on the Cursors Memory button you can fix the setting and use it for the analysis of multiple MST experiments VN ve TEMPER technologies F Update Mode C Users nanotemper Desktop AMP_test amp_test nip Save Curves Curves Background dc uir E X 1 1 1 F user Memory 4 User Manual Monolith NT 115 Date July 2011 Version 03 61 www nanotemper de 49 0 89 4522895 0 VNC Ye TcMPER User Manual technologies 9 2 5 Cold Fluorescence The tab Fluorescence has to be chosen This selection shows the cold fluorescence in each capillary Usually we expect to see an equal fluorescence distribution between the different capillaries but in some cases the cold fluorescence changes with increasing concentration of the titrated molecule If the buffer composition is kept constant this signal als
34. ceeececceeceeeeeseceeeeaecessugeceesenseeessunees 67 11 Typical Thermophoretic Time TACOS nennen ee ssa cala 69 12 Application PROLOCOIS assa sas eai i casco a ia didi ee ee ee 71 User Manual Monolith NT 115 Date July 2011 Version 10 5 www nanotemper de 49 0 89 4522895 0 vo TEMP ES User Manual technologies Safety Considerations To ensure operation safety this instrument must be operated correctly and maintained according to a regular schedule Carefully read to fully understand all safety precautions in this manual before operating the instrument Please take a moment to understand what the signal words WARNING CAUTION and NOTE mean in this manual Safety symbols WARNING A WARNING indicates a potentially hazardous situation which if not avoided could result in serious injury or even death CAUTION A CAUTION indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury It may also be used alert against damaging the equipment or the instrument Do not proceed beyond a WARNING or CAUTION notice until you understand the hazardous conditions and have taken the appropriate steps NOTE A NOTE provides additional information to help the operator achieve optimal instrument and assay performance Read manual label This label indicates that you have to read the manual before using the instrument This label is positioned at the backside of the device Warning symbol
35. d Cursor position Choose the tab Thermophoresis with Jump The Hot Cursors red and the Cold cursors blue are shown in the graph top right on the display Now the fluorescence change after approx 35 seconds of laser heating compared to the initial cold fluorescence is shown Experiment 1st started at 07 0 sf 2 44141 J sf 4 88281 9 2 2 Only Thermophoresis Cursor position VN ve TEMPER technologies Update Mode C Users nanotemper Desktop AMP_test amp_test ntp Zoom On a 7 Cursor Memory The calculated Curve has to be chosen at the tab Thermophoresis The Hot Cursors and the Cold Cursors are shown in the graph top right on the display Distinguish Runs meh aa User Manual Monolith NT 115 Date July 2011 Version 03 Wwww nanotemper de 49 0 89 4522895 0 VN ve TEMPER technologies Update Mode C Users nanotemper Desktop AMP_test amp_test ntp zn ga Cursor Memory 60 VNC Ye TE MPES User Manual technologies 9 2 3 Only Temperature Jump Cursor position The Calculated Curve has to be chosen at the tab Temperature Jump The Hot Cursors and the Cold Cursors are shown in the graph top right on the display Now the fluorescence change that is induced by the mere change of temper
36. d in the manual at 3 2 and 3 3 5 Put the stock on ice and protect it from light 6 Prepare small 16 micro reaction tubes best suited are tubes with a volume of 100ul or less Do not use reaction tubes with higher volume for volumes of 20ul or less This may lead to strong evaporation and may change concentration due to a low volume to surface ratio in the tube Number them from 1 through 16 7 Fill 20ul of the highest concentration you intend to use in the first micro reaction tube number 1 8 Fill 10ul of the buffer you want to use for dilution into the micro reaction tubes 2 to 16 Please note The buffer in tube number one and the buffer in the other tubes User Manual Monolith NT 115 Date July 2011 Version 03 28 Wwww nanotemper de 49 0 89 4522895 0 OONO T MPE User Manual technologies must be the same Otherwise you get a gradient in salt DMSO glycerol or other additives This interferes with the thermophoretic measurement 9 Transfer 10ul of tube number one to tube number two and mix very well by pipetting up and down several times 10 Change the tip of the pipet and transfer 10ul in the next tube Repeat this 15 times and remove 10ul from tube number 16 after mixing 11 Mix 10ul of fluorescently labeled sample with the 10ul of the titrated compound and mix well by pipetting up and down several times Please note that you only have half the concentration you initially had It is more precise to prepare 15
37. e Monolith NT Capillary horizontally into the reaction tube to aspirate the sample Don t touch the capillary in the middle the optical measurement will be performed at this position It is recommended to stop sucking in the liquid when the capillary is filled to 2 3 of its length ta b ni g baz er Be a Position the liquid in center of the Monolith NT Capillary by holding it vertically and shake it after aspirating the sample Leave some air at both ends of the capillary User Manual Monolith NT 115 Date July 2011 Version 03 35 www nanotemper de 49 0 89 4522895 0 OONO T MPzer technologies User Manual Seal the ends with wax Put the capillaries in the slots on the sample tray Note the order of the capillaries Typically the highest concentration is placed in the front of the tray i e closest to the user in the inserted tray This position is denoted as 1 inthe Monolith software 36 User Manual Monolith NT 115 Date July 2011 Version 03 www nanotemper de 49 0 89 4522895 0 VNC Ye TzMPZr User Manual technologies User Manual Monolith NT 115 Date July 2011 Version 03 37 Wwww nanotemper de 49 0 89 4522895 0 OONO MPezer Fr AN U Qies User Manual Place the tray in the instrument by pushing it into the tray slot of the instrument as far as possible The Monolith NT 115 instrument scans the tray and automatically determines the po
38. e like The warranty for all parts supplied and repairs provided under this warranty expires on the warranty expiration date of the original product For inquiries concerning repair service contact NanoTemper after confirming the model name and serial number of your NanoTemper instrument User Manual Monolith NT 115 Date July 2011 Version 10 13 www nanotemper de 49 0 89 4522895 0 VNC OD TEMPEs User Manual technolocles Installation Requirements To ensure operation safety observe the following conditions 1 Do only operate the Monolith NT 115 instrument with the delivered external power supply Power Solve PSG60 12 02 or Sinpro Electronics Power Supply SPU63 105 2 Only connect the external power supply of Monolith NT 115 to an electrical socket containing a protective conductor terminal 3 Ensure that the power plug of the external power supply is well accessible The Monolith NT 115 instrument has to be installed in a way that it does not hinder the access to the external power supply and its power plug Only operate the instrument with the delivered PC Notebook 5 Only operate the instrument with original Monolith NT 115 capillary trays The maximum noise level of the instrument is 64dB A Only operate the instrument in an environment where this noise level is appropriate 7 Operate the instrument in a temperature range of 15 30 C Operate the instrument in a humidity range of O 80 RH If
39. e software automatically saves the capillary scan data and all settings The settings and the scan can be reloaded The Start button starts the Microscale Thermophoresis measurement and all capillaries on the sample tray are measured After pressing the Start button a window appears and the operator is asked to define how often each individual capillary should be measured A repetition means tray is measured once with all capillaries The instrument then repeats the measurement starting at position 1 again Measurement The option delay defines the time between the individual repetitions Also different laser powers can be chosen When analyzing a sample for the first time we recommend 15 40 and 100 laser power User Manual Monolith NT 115 Date July 2011 Version 03 50 Www nanotemper de 49 0 89 4522895 0 vo TE MPES User Manual technologies Fluorescence Display The capillary scan Find Capillaries function as well as the Microscale Thermophoresis data Start function is displayed on the Y Axis of the fluorescence display capillary scan the x axis displays the position mm of the capillaries on the Sample Tray and the y axis the fluorescence intensity 2400 2100 1800 4 8 12 16 20 24 28 32 36 40 44 48 2 56 60 64 68 72 76 Position mm Fluorescence o sa o o o So w a oO oO Microscale Thermophoresis measurements
40. e the MST Report by pushing on the Show Report button A new window appears Add Comment in which you can describe the experiment in a few sentences After pushing the Continue button the MST Report will be generated as an HTML file User Manual Monolith NT 115 Date July 2011 Version 03 66 Wwww nanotemper de 49 0 89 4522895 0 VNC Ye TzMPZr User Manual te CI E U QIES 10 Data Processing of Multiple Experiments The following graphs show experiments where a 150 Da molecule is titrated against a constant concentration of a 50kDa protein The small molecule has an affinity of about 20uM The experiments have been repeated 3 times in the same capillary with the same IR Laser power A response of protein thermophoresis upon addition of the small molecule is observed with an approximate IC50 of 20uM Calculated Curve o O ja oa Concentration If you intend to calculate error bars for this measurement they have to be further processed The individual experiments have a much higher precision than what is observed in the graph above In the following you see the result of each individual repetition Repetition 1 Calculated Curve Hot Cold 10 Concentration User Manual Monolith NT 115 Date July 2011 Version 03 67 www nanotemper de 49 0 89 4522895 0 YN VD MPzEr User Manual technologies Repetition 2 Calculated Curve a O gt pr em 10 Concen
41. ed free dye in your sample it is best to use the NanoTemper labeling kit manual Prepare at least 20ul of sample per capillary to avoid pipetting error and changes in concentration due to sample evaporation You will need approx 5ul to fill a capillary The concentration of the labeled molecule is always kept constant The unlabeled compound is varied in concentration Do not use less than 10 capillaries for an analysis of interaction The observed fluorescence counts of the labeled sample in the respective dilution should be about 80 1500 fluorescence counts when measured with the Monolith NT 115 The concentration of the labeled molecule should be in the range of the expected KD or less The concentration of the titrated compound should vary from about 20x to 0 02x the expected KD 1 Spin down the stocks of the fluorescently labeled molecule and the molecule you intend to titrate in a serial dilution Smin 13000 rpm in a table top centrifuge 2 Check the buffer composition of the labeled compound and the compound that is titrated 3 Prepare a stock solution of the fluorescently labeled molecule at twice the concentration you want to use in the experiment Prepare at least 200ul 10ul x 16 Capillaries 1 5 Test measurements Use the buffer you want to use for determining the affinity 4 Mix the stock well and test the fluorescence intensity in a capillary in the NT 115 instrument and test the sample as suggeste
42. eeneeeeessssssseeeeccccecceeeesaeaaaeassssssseeeeeeeceeeeeeaaaaaaaeaesssses 23 2 7 Dedicated Data Acquisition and Analysis Software ccccsscccccsseccccenseecceuseceeeeecceeeueceessuseeesaeness 23 3 Sample Preparation ceeseseik sinne 24 3 1 Control Kit performing standard CxXpPeriMENt cccccccsssccccesseccccesececceseceeseeceeeeeceeseusecetseners 24 3 2 Selecting the right Monolith NT Capillary cccccccssscccccssecccceseccecesceeceeseceeeeneceeeeneceeseesecetseness 24 Detecting ABEL ER QUO dt 26 3 4 Quick Start Protocol for Sample Preparation ccccccesccccssseccccsseccccescccceeseceeeeeceeseusceeseusecetseners 28 User Manual Monolith NT 115 Date July 2011 Version 10 3 www nanotemper de 49 0 89 4522895 0 OONO T MPE User Manual technologies You should mix small volumes by pipetting up and down To Vortex a sample does not work for small N E a di 29 2 Prepatine EXBELINEN Eee ee meer 30 4 1 Preparing a Molecular Interaction Experiment cccccceseccccssececesececeesececeeeseeeeseecesseneceesegeeees 30 4 2 Preparing an Enzyme Reaction Experiment ccscccscccscccseccscccscctseccscccseetseecseesseeteesseeseeeseeesees 33 6 Monolith Instrument INStructions cccccccccccccssessseecccccesaeesseeeccceessaeeeseeeceeeesseueeseeeseeeessaaeseeeseeeenas 39 Ts Data Acauls LION SOLU IE ee AA Pesa 41 Tok SE CINE Paramete Seen O to 42 7 2 MST assistant The Concentration
43. es 9 3 1 Data point removal Sometimes there are outliers in a data set They can be due to several reasons The first reason is aggregation Take a look at the time trace and if you see a Bumpy signal caused by convective flow of aggregates The second reason is a deviation in concentration Take a look at the fluorescence of the sample if the outlier is also an outlier in fluorescence A third reason for outliers is dirt or water on the outside of a capillary that perturbs the infrared laser The outliers can be removed by the following procedure Click on the data point in the Calculated Curve plot It is highlighted in red Also the time trace is highlighted Curves Orginal Fluorescence Normalized Fluorescence 39 06 2 A 7813 2 156 25 2 3125 2 625 2 1250 2 2500 2 5000 2 10000 2 v Deselect all Extrapolation y o q O a a to a x Hot Cold 102 Concentration In the graph above and in the list of the measurement below this point is automatically marked Remove the check mark of this point for the deactivation User Manual Monolith NT 115 Date July 2011 Version 03 64 Wwww nanotemper de 49 0 89 4522895 0 VN Ye TEMPER User Manual technologies Orginal Fluorescence Normalized Fluorescence nA gt oO 39 06 2 A 7813 2 156 25 2 3125 2 625 2 1250 2 2500 2 5000 2 10000 2 v 8 Ll
44. gly absorbed by water Before the IR Laser is switched on a homogeneous fluorescence distribution Fcold is observed inside the capillary When the IR Laser is switched on two effects separated by their time scales contribute to the new fluorescence distribution Fhot A temperature jump T jump whose relaxation time is fast 50 ms and the thermophoretic movement of the molecules on the slower diffusive time scale 10 30 s Both the milliseconds fast temperature jump and the thermophoretic movement can be used to characterize the molecules and to quantify the binding affinities While the mass diffusion D determines the kinetics of depletion the Soret coefficient Sr determines the steady state concentration ratio Chot Ccoig eXP St AT 1 S AT under a temperature increase AT The normalized fluorescence Frorm Fhot Feoig Measures mainly this concentration ratio in addition to the temperature jump OF OT In the linear approximation we find Frorm 1 OF OT ST AT By denoting x the fraction of labeled molecules A bound to their targets T the changing fluorescence signal during the titration of target T is given by Frorm 1 x Frorm A x Frorm AT Where Frorm A is the normalized fluorescence of only unbound labelled molecules A and Frorm AT is the normalized fluorescence of complexes AT of labeled molecules bound to their targets in saturation every labeled molecule bound to a target User Manual Monolith NT 115 Date July 2011 Vers
45. h pose a risk of infections User Manual Monolith NT 115 Date July 2011 Version 10 8 www nanotemper de 49 0 89 4522895 0 VNC Ye TEMPER User Manual technologies Regulatory Statement The following safety and electromagnetic standards were considered e IEC 61010 1 2001 Safety requirements for electrical equipment for measurement control and laboratory use Part 1 General Requirements e EC 61010 2 010 2005 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 010 Particular requirements for laboratory equipment for the heating of materials e IEC 61010 2 081 2001 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 081 Particular requirements for automatic and semi automatic laboratory equipment for analysis and other purposes e IEC 60825 Laser safety e IEC61326 1 2006 EMC Electrical equipment for measurement control and laboratory use EMC requirements e IEC 61000 3 2 2006 EMC Limits for harmonic current emissions equipment input current up to and including 16A per phase e IEC61000 3 3 2008 EMC Limits User Manual Monolith NT 115 Date July 2011 Version 10 9 www nanotemper de 49 0 89 4522895 0 VN ve MPzr User Manual technologies Specifications Input external Power Supply 90 264 VAC 10 47 63 Hz 230 VA max Output external Power Supply 12 VDC 5 0 A max 2 4 A typical Electrical Input Mono
46. he measurement data can be chosen and an extra window appears User Manual Monolith NT 115 Date July 2011 Version 03 57 Wwww nanotemper de 49 0 89 4522895 0 VO TzMPEr User Manual technologies Experiment 002 started at 27 05 2011 02 20 31 24 Curves LED Power 25 Distinguish R Use Average Hot Cola Tremor Choose one or multiple runs Evaluation Points User Manual Monolith NT 115 Date July 2011 Version 03 58 www nanotemper de 49 0 89 4522895 0 VN Ye TEMPER User Manual technologies Here you see the curve for the selected measurement in the picture above NanoTemper Analysis 1 2 10 Runs Orginal Fluorescence Normalized Huorescence Cycles oO oO oO co oO co co oO a E 5 c lt 7 lt o E 7 o Concentration 9 2 MST data analysis Following analysis can be performed Only Thermophoresis only Temperature Jump Thermophoresis with Jump Cold Fluorescence Hot Cold Hot Cold Themophoresis eee o ee eaeSaeSeoerc In the following capital all types of analyses are explained User Manual Monolith NT 115 Date July 2011 Version 03 59 Wwww nanotemper de 49 0 89 4522895 0 User Manual OONO T MPzer technologies 9 2 1 Temperature jump and Thermophoresis Standar
47. ing from standard and proprietary buffers to complex bioliquids including blood serum cell lysates and the like 2 7 Dedicated Data Acquisition and Analysis Software The Monolith NT 115 is supported by a software for data acquisition and analysis User Manual Monolith NT 115 Date July 2011 Version 03 23 Wwww nanotemper de 49 0 89 4522895 0 VN Ve T MPZr User Manual Cf hnoloaies 3 Sample Preparation For detailed information about handling capillaries please read chapter 5 3 1 Control Kit performing standard experiments Control Kits containing standardized sample material are recommended when using the Monolith NT 115 instrument for the first time for monitoring the correct performance of the Monolith NT 115 for training of new lab employees Optimized Control Kits for use in Microscale Thermophoresis are available either with red fluorescent or with blue fluorescent labeled bio molecules The kits contain a fluorescent labeled DNA molecule unlabeled binding partner and buffer C030 Monolith NT Control Kit RED Molecular Interaction Control Kit for Monolith NT 115 BLUE RED or Monolith NT 115 GREEN RED C031 Monolith NT Control Kit GREEN Molecular Interaction 5 Control Kit for Monolith NT 115 BLUE GREEN or Monolith NT 115 GREEN RED C032 Monolith NT Control Kit BLUE Molecular Interaction Control Kit for Monolith NT 115 BLUE RED or Monolith NT 115 BLUE GREEN 3 2 Selecting the right
48. ion 03 53 Wwww nanotemper de 49 0 89 4522895 0 VNC Ye T MPE E User Manual technologies Basic setup of the instrument LED excitation Fluorescence detector IR Mirror 4 o e seen Prq rt Back State MST T Jump diffusion Q no molecule flow O pt j cs E ehren Backdiffusion RS ha Kemer Temp Jump capill Ex Thermo phoresis 0 9 ra Normalized Fluorescence 0 8 1 0 40 50 IR Laser on Time s IR Laser off The dissociation constant K4 quantifies the equilibrium of the reaction of the labelled molecule A concentration ca with its target T concentration cr to form the complex AT concentration car and is defined by the law of mass j Ca XE action as K A T CAT where all concentrations are free concentrations During the titration experiments the concentration of the labelled molecule A is kept constant and the concentration of added target T is increased These concentrations are known and can be used to calculated the dissociation constant The free concentration ofthe labelled molecule A is the added concentration minus the concentration of formed complex AT The same holds for the target T Thus the Kg is a c car x ed car d CO ge By solving this quadratic equation for the fraction of bound labelled molecules the results from the titration series varying while is constant can be fitted with the following equation to determine Kg
49. ion for the capillary scan can be selected to find the capillaries When pressing the Find Capillaries button the Monolith NT 115 automatically scans the sample tray and measures the fluorescence at predefined positions i e defined by start end value Capillaries containing fluorescently labeled molecules result in fluorescence peaks on the Fluorescence Display When the Find Capillaries procedure is completed a window appears showing the number of the capillaries on the tray Capillary No 1 is the one on the tray close to the operator NOTE The Find Capillaries procedure should not be cancelled otherwise the capillary positions are not stored When the capillary finding procedure is used to check for the protein fluorescence intensity the Find Capillaries procedure can be cancelled the LED power adjusted and the Find Capillaries procedure function restarted User Manual Monolith NT 115 Date July 2011 Version 03 48 www nanotemper de 49 0 89 4522895 0 VNC Ye T MPES User Manual technologies Display of the fluorescence of each capillary on the tray Fluorescence 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 Position mm Note As can be seen in the figure above in some cases the fluorescence of a sample increases or decreases with increasing concentration of binding partner The change in fluorescence is regularly observed upon interaction of two molecules
50. lith NT 115 instrument 12 VDC 5A Environmental Operating temperature 15 30 C Indoor use only Storage temperature 20 30 C Humidity 0 80 noncondensing Operating altitude max 2000m Monolith NT 115 Size width 33 cm 13 height 45 cm 17 5 depth 51 cm 20 Weight 22 kg 51 Ibs net Power Supply Size width 10 6 cm 4 2 height 3 cm 1 2 depth 6 5 cm 2 6 Power Supply Weight 0 23 kg 0 51 Ibs net max IR Laser Wavelength 1475 nm 15 nm Power 120 mW max Monolith NT 115 Laser classification Device is LASER PRODUCT CLASS 1 Temperature Control Range 20 C 50 C at Room Temperature 25 C Accuracy 0 3 C Noise Level of Monolith NT 115 instrument max 64 dB A User Manual Monolith NT 115 Date July 2011 Version 10 10 www nanotemper de 49 0 89 4522895 0 VNC Ye TEMPER User Manual technologies Connections All ingoing and outgoing connections can be found at the rear panel of the instrument Ethernet USB Power Switch DC Input Ethernet Connector for connecting the Ethernet cable to the PC Notebook USB Connector for connecting the USB cable to the PC Notebook Only used for service by NanoTemper service Power Switch When the switch is pressed on position I the instrument is switched on DC Input The connector to the external power supply Connect with the external power supply User Manual Monolith NT 115 Date
51. most measurements the room temperature is sufficient so there is no adjustment necessary During the measurement the display show the acitivity of the optical components Light Emitting Diode LED for Fluorescence excitation and LASER for the activity of the IR Laser for local sample heating LED OFF LED ON and LASER OFF LASER ON User Manual Monolith NT 115 Date July 2011 Version 03 40 Wwww nanotemper de 49 0 89 4522895 0 VNC YD MPzer User Manual technologies 7 Data Acquisition Software With the Monolith Data Acquisition Software Interface all necessary parameters for the Monolith NT 115 can be set It does not matter whether you switch on the Monolith NT115 or the data acquisition software first After starting the Data Acquisition Software from the desktop the following screen appears File Settings Info Connection Status Ready RAR LED LASER ai Concentration Finder LED Selection Blue x nis s Laser Power E TEMPER o cura bem Concentration technologies LED Power mja 20 x Final Laser Off Time 5 s E i Br no Temperature Control Gate Capillary Scanning N Experiment Project Current Temp 26 0 C Stat l z E Switch On _ NewProject Set Temp dE gt 25 C End 6 a E i LoadProject Open Close Find Capillaries ii Experiments Name
52. mper de 49 0 89 4522895 0 OONO T MPE User Manual technologies In order to simulate how the binding curve will look like please enter the following values No of Capillaries 1 16 Concentration cA enter the concentration of the fluorescently tagged molecule Maximum of Concentration cT enter the highest concentration of the unlabeled molecule Dilution Usually we recommend doing 1 1 dilution of the unlabeled titrated molecule Kd Expected dissociation constant kD Interval tool In order to use the kD Interval tool please click on the kD Interval box Enter the kD range for example between 10 1000 nM and push the Optimise for Kd Interval button The software adjusts automatically the concentration of the labeled molecule cA and the concentration range of the unlabeled molecule cT 16 No of Capillaries oe 25 0 concentration CA ici VNC TO 40000 0 Maximum of Concentration CT E Kd Max TzMPEZErRr 11 v Di ien 100 10000 technologies Cap E Number i Concentration Find a Proper Concentration Distribution 40000 00 mn Kd m max Kd 20000 00 10000 00 5000 00 2500 00 1250 00 625 00 312 50 156 25 78 13 __ 39 06 19 53 9 77 4 88 2 44 122 ico Nm on a win Oo 3 o 2 o 3 o m _ Iw N m O a ema emb in a 10 Concentration User Manual Monolith NT 115 Date July 2011 Ver
53. ned The equilibration time of Microscale Thermophoresis depends on molecular diffusion and is in the order of 10 30 seconds However in some reactions a result is obtained in the fast temperature jump in this case measurement times of 1 to 5 seconds are sufficient to measure the affinity a value of 30 seconds is recommended as a starting point for an unknown sample With the Laser Power the heating power and thus the strength of the Microscale Thermophoresis can be varied between 10 to 100 i e 0 5V and 2V When analyzing an unknown system it is recommended to check 20 40 and 80 Laser power NOTE To obtain optimal results with the Monolith instrument it is strongly recommended to test a wide parameter space in sense of laser power Temperature Control Temperature Control Current Temp 24 2 C Set Temp Em 30 C With the Set Temperature the temperature of the capillary tray capillaries can be varied When pushing the Switch Off button the temperature of the capillaries will be set to the preset temperature The current Temperature shows the actual temperature of the capillaries For the most measurement the room temperature is sufficient so there is no adjustment necessary If you use the temperature control equilibrate the capillary tray including the filled capillaries at least for 5 minutes before starting the MST Analysis To achieve best results the capillary positions at the ends sh
54. noTemper software program has failed causing an error or improper operation this may be caused by a conflict from another program operating on the Notebook PC In this case take corrective action by uninstalling the conflict product s 7 NanoTemper is a registered trademark of NanoTemper Technologies GmbH in Germany and other countries User Manual Monolith NT 115 Date July 2011 Version 10 12 www nanotemper de 49 0 89 4522895 0 TEMP ES User Manual technologies Limited Warranty Products sold by NanoTemper unless otherwise specified are warranted for a period of one year from the date of shipment to be free of defects in materials and workmanship If any defects in the product are found during this warranty period NanoTemper will repair or replace the defective part s or product free of charge THIS WARRANTY DOES NOT APPLY TO DEFECTS RESULTING FROM THE FOLLOWING IMPROPER OR INADEQUATE INSTALLATION IMPROPER OR INADEQUATE OPERATION MAINTENANCE ADJUSTMENT OR CALIBRATION UNAUTHORIZED MODIFICATION OR MISUSE USE OF UNAUTHORIZED CAPILLARIES AND CAPILLARY TRAYS USE OF CONSUMABLES DISPOSABLES AND PARTS NOT SUPPLIED BY AN AUTHORIZED NANOTEMPER DISTRIBUTOR 6 CORROSION DUE TO THE USE OF IMPROPER SOLVENTS SAMPLES OR DUE TO SURROUNDING GASES 7 ACCIDENTS BEYOND NANOTEMPER S CONTROL INCLUDING NATURAL DISASTERS SS TH This warranty does not cover consumables like capillaries reagents labeling kits and th
55. o reflects a binding event This is readily explained by a change in the electrostatic surrounding of the dye that changes the fluorescence intensity by shifting the fluorescence intensity measured with the Monolith detector 9 3 Curve Fitting If there are enough data points the curve can be fitted The program calculates a fit according to the law of mass action Load an experiment b a 910 o oO o o q Thermophoresis with no Jump 10 Concentration In the field Concentr on the right side type the concentration of the constant fluorescently labeled molecule in the same order of magnitude that is used on the x axis e g nM and mark the Checkbox Fix Hot Cold Thermophoresis Thermophoresis with Jump Temperature Jump Fluorescence Hot Cold Evaluation Points ES Ga Gold Lenath 5 Themophoreat ze i E 7 Li w b on fi Ji ji 1000 00 S1 Bode 900 00 Kd Fit Hill Method Kd Border Hill Border Thermophoresis with no Jum co to oO oo co on 250 Ka Concentr Unbound Bound Concentration Fix V Fix Fix Fix User Manual Monolith NT 115 Date July 2011 Version 03 62 Wwww nanotemper de 49 0 89 4522895 0 vv MPEs User Manual technologies 1 Push the Complete button Hot Cold Thermophoresis Thermophoresis with Jum
56. ons so and mix thoroughly and fast Prepare the Samples fast on ice Transfer both samples in to a capillary see below and start the measurement using the Monolith analysis software and choose the number of repeats and a delay time O fastest repetition NOTE If you want to check if the enzymatic reaction is specific or you want to increase signal strength you can add a molecule into the sample that specifically binds to the product This way the enzyme kinetic is detected indirectly If the fluorescence intensity is too high even at the lowest LED power gt 2500 counts you can dilute the fluorescently labeled substrate with unlabeled substrate User Manual Monolith NT 115 Date July 2011 Version 03 33 Wwww nanotemper de 49 0 89 4522895 0 VN Ye amp T MPrerR User Manual technologies Alternatively you can choose a different LED Detector combination that is not optimal for your dye e g Blue fluorescence excitation wavelength and Cy3 detector User Manual Monolith NT 115 Date July 2011 Version 03 34 Wwww nanotemper de 49 0 89 4522895 0 VN Ye TzMPZrR User Manual technologies 5 Sample Loading Be careful when using the glass capillaries Broken capillaries are harmful to skin and eyes Discard the capillaries only in waste boxes that are intended for use of glass waste Glass capillaries are not intended for use with infectious samples Wear gloves safety glasses and lab coat Stick th
57. ould not be used User Manual Monolith NT 115 Date July 2011 Version 03 44 Wwww nanotemper de 49 0 89 4522895 0 VNC Ye TcMPER User Manual technologies 7 2 MST assistant The Concentration finder tool This tool will help you to design an appropriate MST Experiment In order to calculate the KD of an interaction it is important to chose an optimal concentration range of the analyzed molecules You can either simulate how your binding curve will look like at known KD or alternatively you can use the Kd Interval tool if you only know the binding affinity range The tool will help you to choose an optimal concentration of the fluorescently labeled molecule and will determine the best concentration range of the unlabeled molecule Concentration Finder Concentration After opening the Concentration finder tool the following screen appears 16 No of Capillaries 50 0 Kd Optimise for Kd Interval 500 Concentration CA pes VNC nO uni Inu TEMPER Em 1000 0 10000 0 technologies ES a Persie Find a Proper Concentration Distribution gt i 5000 00 numerical 2 2500 00 3 1250 00 A 625 00 3 312 50 6 156 25 E 7 78 13 8 39 06 Neca ed 9 19 53 Tv 3 13 5 E 10 9 77 a 11 4 88 m 12 244 13 1 22 14 0 61 15 0 31 16 0 15 102 1071 10 10 102 10 104 10 a Concentration User Manual Monolith NT 115 Date July 2011 Version 03 45 Wwww nanote
58. p Temperature Jump Huorescence Hot Cold Evaluation Points O LED 25 Laser 17 8 see egg oo a o A A Thermophoresis with no Jump co wo oO co co on Concentration Fix Push the Fit Curve button After this you obtain a curve that fits your results best Hot Cold Thermophoresis Thermophoresis with Jump Temperature Jump Huorescence Hot Cold Evaluation Points Fitted Curves p to O o a N N S oO in oO on oO w oO oO co co or Y E 3 5 o E 3 2 8 905 o a o E o 890 Kd Fit Hill Method Kd Border Hill Border A 118000 250 922 61 891 40 10 Ka Concentr Unbound Bound Concentration Fix V Fix Fix Fix The Dissociation constant is shown in the Kd Fit field Note The values for Unbound Bound and Diss Const can be changed as well Further you can choose different fit modules by pushing the tabs Simple Kd or Hill Method The preview function will plot the graph corresponding to the actual settings without fitting i e adjusting the values Pushing the Border tab allow to define the concentration range for fitting This can be used in case of biphasic binding events User Manual Monolith NT 115 Date July 2011 Version 03 63 Wwww nanotemper de 49 0 89 4522895 0 YN VED T MPzEr User Manual technologi
59. rt If you move the instrument alone it poses a risk of personal injury or damage to the instrument CAUTION The manual opening of the instrument is not allowed Manual opening pose a risk of personal injury or damage to the instrument Contact NanoTemper service personnel if you need to open the instrument CAUTION The instrument contains an IR laser module invisible laser radiation class 3B according to IEC 60825 1 2007 Lasers or laser systems emit intense coherent electromagnetic radiation that has the potential of causing irreparable damage to human skin and eyes The main hazard of laser radiation is direct or indirect exposure of the eye to thermal radiation from the infrared B spectral regions 1 400 nm 3 000 nm Direct eye contact can cause corneal burns retinal burns or both and possible blindness Do not open the instrument while it is switched on Manual opening of the instrument during operation poses a risk of personal injury or damage to the instrument When the instrument is used as intended it emits laser radiation of LASER CLASS 1 CAUTION Mechanical moving parts within the instrument can pinch or injure your hands or fingers Do not touch the instrument while parts are moving CAUTION The instrument contains a temperature regulator to control the sample temperatures Some accessible parts of the instrument can reach temperatures up to 50 C Avoid to touch the temperature controlled parts of the instrument for a longer
60. sion 03 46 www nanotemper de 49 0 89 4522895 0 VN Ye TEMPER User Manual technologies 7 3 Concentration Input Use the Dilution bar and select the appropriate dilution depending on the prepared dilution for example 1 1 Type the highest concentration of the unlabeled binding partner at position No 1 No 1 is the first capillary from the operator s point of view and typically the highest concentration is placed in front After marking the first concentration move while holding the left mouse button to the next concentration field below The concentration will be calculated automatically Please use only numbers in the Concentration column The Name column accepts both numbers and letters You can add information in this column like Nanomolar etc oo s a E _ Ba 195 31250 97 65625 no asse O m Bo 12 20703 MeasurementNo 1 ER Dilution 11 1 v From 1 J 3 I User Manual Monolith NT 115 Date July 2011 Version 03 47 Wwww nanotemper de 49 0 89 4522895 0 VNC VD TEMPER User Manual technologies 7 4 Instrument Control Functions Open Close Gate The Open Close button is deactivated by the default software settings To open or close the device press the button on the touchscreen See chapter 6 Find Capillaries Capillary Scanning Start 1 v End 16 x Find Capillaries The start and end posit
61. sition of the capillaries on the tray Capillary No 1 is the one on the tray close to the operator see Software section for details User Manual Monolith NT 115 Date July 2011 Version 03 38 Wwww nanotemper de 49 0 89 4522895 0 ONNO T MPzer User Manual technologies 6 Monolith Instrument Instructions First start the Monolith NT at the back side After 15 seconds the display switches on automatically LED OFF LASER OFF E 250 EM Temperature 24 4 C Before starting a measurement it s necessary to start the Data Acquisition Software If there a connection between the Monolith NT and the Software the following symbol is shown in the upper left of the display With Open Close the instrument is opened to put in the sample tray with the capillaries When pushing Open Close again the flap closes When pushing on the off symbol that changes to On the temperature of the capillary tray can be increased or decreased by using the arrow upwards and User Manual Monolith NT 115 Date July 2011 Version 03 39 www nanotemper de 49 0 89 4522895 0 VNC Ye T MPES User Manual technologies arrow downwards symbol Please note this is not the laser induced temperature used for the measurements It adjusts the overall capillary temperature i e ambient temperature The temperature of the capillaries is shown in the display between the two arrows For
62. tration Repetition 3 Calculated Curve Hot Cold 10 Concentration However the baseline of each individual experiment is shifted to lower values This is for example due to a change in the initial conditions due to photo bleaching of the sample A procedure to average different experiments consists of the following steps 1 Export the results of the repetition measurement to a txt file And open it with a software like Microsoft Excel 2 Substract the baseline from each individual experiment e g 883 880 and 878 from repetition 1 2 and 3 respectively 3 Divide each individual repetition by its amplitude i e difference between unbound and bound state You obtain the amplitude by fitting the experiment using the Monolith Software 4 The Y Axis of the resulting graphs can now be interpreted as Fraction bound You can now average the traces and calculate the error bars User Manual Monolith NT 115 Date July 2011 Version 03 68 www nanotemper de 49 0 89 4522895 0 OONO T MPE User Manual te CI E U QIES 11 Typical Thermophoretic Time Traces The most prominent shape of MST time traces of a titration series is a signal that shows a continuous decrease of fluorescence intensity during However there are also very different shapes observed These may be a result of thermophoresis from lower to higher temperatures or temperature jump and thermophoresis that lead to a change in fluorescence
63. ul of the titrated sample transfer 10ul to fresh tubes and then mix with 10ul of the labeled molecule 12 Incubate the sample at conditions of your choice before filling it into the capillaries Incubation temperature and time can differ between different molecules In most cases 30 60 minutes incubation at room temperature are sufficient NOTE You should mix small volumes by pipetting up and down To Vortex a sample does not work for small volumes User Manual Monolith NT 115 Date July 2011 Version 03 29 Wwww nanotemper de 49 0 89 4522895 0 ANOO T MPEs User Manual technologies 4 Preparing Experiments 4 1 Preparing a Molecular Interaction Experiment For performing a molecular interaction experiment and measuring the dissociation coefficient KD a titration series of up to 16 dilutions is prepared where the concentration of the fluorescent binding partner is kept constant and the concentration of the unlabeled binding partner is varied Do not use less than 10 capillaries NOTE Also competition experiment can be performed to analyze interactions E g a complex between two proteins is formed one of them fluorescently labeled The concentration of this complex is kept constant and a small molecule or any other competitor is titrated to this complex When the small molecule is capable of separating the proteins it is observed in thermophoresis Thus a functional assay is performed Prepare the labeled

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