Home

ITC Expert User`s Manual

image

Contents

1. Simulation Macromolecule Conc 0 0040 mM Maximum Macromolecule Conc 3mM e i e E e Minimum Macromolecule Conc 0 01 mM e mg Optimal Macromolecule Conc 0 1 mM Fi a S P m vE 4 B J f ka E a f G eo __ Ea am E a E E O E r 0 0 0 5 1 0 1 5 2 0 Molar Ratio Exporting To an INJ File Once you are satisfied with your experiment s design the simulation parameters can be exported as an INJ file An INJ file is an Experimental Parameters file used by VPViewer To export to an INJ file simply click on the Export to INJ File button 11 Once the button 1s clicked a Save INJ File As dialog box will open Simply name the INJ file and click Save The parameters can then be loaded into VPViewer by clicking Load Run Parameters and choosing the appropriate INJ file Save As Save in SETUP amp ek E B SAMPLE INI lastrun Linj File name SAMPLEN i Save as type IN Cancel Help Comments Additional Controls Binding Models ITC Expert can simulate either One or Two Site models To activate the Two Site model click on the Two Set of Sites switch 12 ITC Binding Models a One Set of Sites x Two Set of Sites The controls will then show N K and DH control boxes for each site First Site Mf Sterchiametry K EAM DH Cal Mol 1E3 The Two Site model will then be plotted Si
2. 3 mM Minimum Macromolecule Conc 0 01 mM O Optimal Macromolecule Conc 0 1 mM 4 eg aa AH KCal mole of Injectant 0 0 0 5 1 0 tS 2 0 Molar Ratio When Heats Are Too Low We will define a good ITC experiment so that its isotherm has a C value between 10 and 3000 the total heat of the experiment is at least 50 uCal and the first injection is at least 5 uCal During the simulation the total heat of the experiment is measured If the total heat is less than 50 uCal the concentrations are increased If the first injection is less than 5 wCal the number of injections is decreased Below is a simulation of an extreme case The binding parameters for this simulation were N of 1 K of 10 M and a DH of 150 Cal Mol Deg When computing the isotherms at Cs of 10 minimum conc and 100 optimal conc the total experimental heats were too low In raising the concentrations of those curves to a minimal heat level the 2 curves converge In this simulation the minimal concentration and optimal concentration are the same 0 25 mM which corresponds to a C of 249 Concentrations lower than that do not generate enough heat 0 02 Simulation Macromolecule Conc 0 25 mM Maximum Macromolecule Conc 3 mM Minimum Macromolecule Conc 0 25 mM 0 00 o aee gt pe e Optimal Macromolecule Conc 0 25 mM F A 0 02 0 04 0 06 0 08 0 10 0 12 AH KCal mole of Injecta
3. a component of each experimental injection If the heat of the reaction is large then slight mismatch between the ligand solution and the cell solution may not affect the experiment and fitted parameters If the heat of the reaction is relatively low as 1s often the case then the heat of dilution and sample solution matching are critical The heat of dilution arises from mixing of the two components ligand and macromolecule solutions in the ITC sample cell As an injection is made into the sample cell friction force of the ligand injecting into the cell slight temperature mismatch between the ligand and macromolecule solutions and any mismatch of the ligand and macromolecule solutions will yield a heat known as the heat of dilution Each injection will have a heat of dilution associated with it The heat arising from friction and temperature mismatch will change depending on the volume and rate of the injection The heat of component mismatch is purely a function of sample preparation and solution match Solution mismatch has been found to account for the majority of the heat associated with the heat of dilution Solution mismatch is especially troublesome when the heats of the reaction are low working at low reactant concentrations or near the temperature at which the heat of the reaction 1s zero Therefore it is very important to prepare experimental samples such that the solutions of each are as closely matched as possible Matching of the two
4. used the temperature dependence of binding heats to optimize experiments and obtain additional structural information associated with binding reactions Most binding reactions have a temperature at which DH 0 If you collect data within 5 to 10 degrees of this temperature then the heats are almost always going to be low If you change the temperature of the experiment then you will increase the heat of the reaction and obtain better signal to noise Interestingly many systems have values for DH that go through zero between 20 and 30 degrees Section 4 Troubleshooting Why is my Isotherm too steep Most likely during experimental design K was underestimated If the isotherm is indeed that steep C must be in excess of 3000 This would mean that K was underestimated by at least a factor of 30 if C was set to 100 Retry the experimental design wizard using a value of K 30 times greater However once the simulation curve is plotted use minimize sample button to set C to 10 This will give even more range of K values up to 300 and rerun the experiment Why does my Isotherm not look like a Sigmoid Most likely during experimental design K was overestimated C in this case is below 10 K was overestimated by at least 10 Retry the experimental design wizard using a value of K 10 times greater Then set C to 3000 This will give even more range of K values up to 300 and rerun the experiment 18
5. MicroCal ITC Expert User s Manual SECON ls EC Expert ts al Or Oui sis otic E EEA EE EEA cues Mansa ses oa nes ae 3 Mima Hcatsand Tae MOMS 3 380t2c 8 aon stadatits iiodnetasasaiaiatebackatgaa tite R 3 OM irs 6 lt Beene Nee gee ne ee a ao eo ne Se eee 3 DETU TIa ESA IN rey EIRE EIA an ont ee ery Pek ed ere eet a enon eer Pee eo eee eet A I AE bet eo eee EIA AE ee 4 sie E een a AT eer eee eae et Samy ner EAN EIS Ee en mea EO TER RN Caer Stee erie Conn eer Ean EE A mt Conn ere 4 LOW Roemer re Rec mne er nee te cne me emer eer Meee et eee mC eT ee erm a mn ee nT Ce meme mere eer meer eT 6 Concentrations IRA Ox sminka a E ar e E a e R T 6 Section 2 IMEC EXPE OnTO eaa a A 7 The Experimental Design Wizard Walkthrough cccseesssseeeeeccceceeeeeeeeceaaaeeeeessssseeeeeeceeeeeseeseeeaeaasaaeeesseeeees 7 Sarino Ee WAST Oar ch er a E E T E T ease ease ee a 7 Epon mental hy IN AIC Sates oats ans E T E E TOENE O N E TN E 8 When Heats are OOO can ccna etr xis e AE A E bs iion E vote anus no suas OE 9 ACI MISHIVS NSS MMU ALCO CUVE ea duedalnidel chitotceasdadiucedide dyedaenidel dito tceasdaiiuce idedvehsbeiduectieetee 10 Adjustines the Simulation Out of Rance lsieni uea a adaa aeaa nied aaa 11 Exporting TO AMINI TIE cecen a E A E ERE T A oem aoows 11 Vas OU e sees kE ONTOS esre a E E N 12 Brame MOda cae cia E ea ahs saute TATAE EEEE ETE E ied en be Resneveides ee awe 14 BOUON eee eee en ee Cee Cerne eRe nearer renee CTE CERT en e
6. Parameters in Plot This switch will past the binding parameters and experimental parameters to the simulation plot AH KCalimoale of Injectant 0 0 0 5 1 0 15 20 Molar Ratio Constant C or Constant Concentrations Since C is proportional to K and concentrations when adjusting K either C or concentrations must be kept constant Keeping C constant will keep the shape the constant while changing K When keeping concentrations constant the shape of the curve will change 15 Section 3 Sample Considerations Sample Preparation Recommendations Care in preparation of samples for use in the Isothermal Titration Calorimeter ITC is critical to the overall quality of the binding experiment Three aspects of the preparation are of particular importance matching of component buffers concentrating the experimental samples and accuracy of the input sample concentration Attention to these three aspects of the binding experiment will ensure the greatest chance of success Lack of attention to these aspects of the binding experiment may lead to large errors in fitted parameters and failure of the experiment Sample Solution Preparation When considering the components of the heat measured during an ITC binding experiment one must remember the heat of dilution and compare these heats to the total heat of the reaction The heat of dilution 1s the heat associated with injection of the ligand solution into the cell buffer The heat of dilution is
7. at will simulate and help to design a One Site or Two Site ITC binding experiment The program will use input guesses N K and DH to calculate the proper concentrations number of injections and injection volumes Through its algorithms the wizard will ensure that the ITC experiment produces at least 50 uCal of total energy a minimum of 5 uCal of energy for the first injection and a desirable C value and binding isotherm shape that can be easily fit to the calorimetric model Once the simulation passes the necessary heat requirements the isotherm is plotted in the simulator The example below describes a One Site binding model Starting the Wizard To start the wizard click on the Start Experimental Design Wizard Button New une 1 i Fa L i a This will open the Binding Parameters dialog box You must enter guesses of N K and DH to initiate the wizard Binding Parameters Cancel Nil K 1000000 DH 15000 The wizard will begin by setting C to 100 and solve for the macromolecule concentration The program will then determine the appropriate number of injections and injection volumes for the experiment and plot the isotherm hh C3 AH KCal mole of Injectant Molar Ratio The experimental parameters are then displayed in the parameter control boxes Cell Concentration mith 0 100 Syringe Concentration mM 1 0 ey Number of Injections 25 m Injection Vclume uL 7 00 E C Sigmoidis
8. be filtered to remove any particulates introduced during the concentrating process Use of a 0 45 M filter will usually suffice It is always a good idea to visually inspect samples before you load them into the ITC This is best accomplished when the sample is in the glass loading syringe If particles or aggregation is evident in the syringe then the solution should be filtered before loading the cell This step can save sample material and prevent experiment failure Sample Concentration Measurement In an ITC binding study the concentration of each species must be well known to obtain accurate values for stoichiometry binding affinity and normalized heats or the enthalpy of binding DH in kcal mol Error in the concentration of macromolecule in the cell will directly affect the stoichiometry while error in the concentration of ligand will affect all three fitted parameters of the binding experiment Ka n DH The two procedures most often used in sample concentration determination are the weighing of dry sample and dissolving in a known volume of solution and direct spectroscopic measurement Dilution of a stock solution of known concentration is often used to prepare samples since concentrated stocks are more readily prepared than dilute solutions Each method may provide an accurate measure of concentration however it is important to recognize error associated with each The error in sample preparation and concentration measurement will lar
9. components for an ITC experiment is typically accomplished by dialysis of the two components in the same solution until each component solution is identical Dialysis is preferred over other methods and is standard in protein sample preparation However many experiments are performed using small molecules for ligand which may be difficult to dialyze Another problem may arise if solution components are used that do not dialyze well viscous solutes In these situations a pure dry sample is diluted into the experimental buffer For a small molecule protein titration pair the recommended method is extensive dialysis of the protein component and subsequent dissolving of the pure small molecule in the final dialysis buffer Preparation of viscous non dialyzing solutions requires that the samples be pure before dissolving in the solution Protein samples should be dialyzed against water if possible and then lyophilized The pure lyophilized protein may then be dissolved in the solution Small molecules may be purified with chromatographic methods and subsequently dried Caution must be taken with molecules that are purchased from a supplier since the solute components and their final concentrations may not be known One final consideration when preparing the component solutions for ITC is solution pH The pH of each component solution must be matched to avoid additional heat from pH equilibration The greater the difference in pH between the two components
10. etails of the experimental and sample conditions so that they can be applied to a real experiment with the VP ITC instrument Minimal Heats and Injections To confidently fit the binding isotherm the experiment must meet the following conditions e Total heat of experiment is at least 50 wCal e Number of injections must be 10 or greater e The heat of the first injection must not be lower than 5 uCal C Parameter The critical parameter which determines the shape of the binding isotherm is the unitless parameter C which is the product of the binding constant K times the total macromolecule concentration in the cell at the start of the experiment M times the stoichiometry parameter N C K M N Very large C values lead to very tight binding and the isotherm is nearly rectangular in shape with the height corresponding exactly to DH and with the sharp drop occurring precisely at the stoichiometric equivalence point N in the molar ratio Note DH represents AH throughout manual The shape of this curve is invariant with changes in K so long as the C value remains above ca 5000 As C is reduced by decreasing M 1 e holding K DH and other parameters constant the drop near the equivalence point becomes broadened and the intercept at the Y axis becomes lower than the true DH In the limit of very low initial M concentration cf C 0 1 the isotherm becomes featureless and traces a nearly horizontal line indicative of very weak binding It is a
11. gely depend on analytical technique of the user Temperature Dependence of Binding Heats The temperature dependence of binding heats is a phenomenon often overlooked by users of ITC Consequently experimental studies may be abandoned when investigators measure the binding enthalpy of a reaction at a temperature where the heat of binding is near zero In such a case the signal to noise ratio 1s poor the binding isotherm is not well defined and the heats of dilution are comparable to the reaction heats The investigator may conclude that ITC is not suitable to study the interaction of interest On the contrary the temperature dependence of the heat of reaction is a valuable tool for optimizing the study of any binding event The heat of binding for a given reaction and the enthalpy change DH is typically temperature dependent As you change the temperature of an experiment the raw injection heat and therefore DH change as well The temperature dependence is due to the heat capacity change of the event DCp DH T The value of DCp for biological interactions is almost always negative and ranges from approximately 0 3 to 2 kcal degree mole If 17 you collect a series of experiments using the same binding partners at different temperatures and plot the fitted values of DH vs temperature then the slope of a straight line through the data is the DCp DCp ACp The DCp may be used to obtain higher heats of reaction and therefore better data Fo
12. ity Experimental Boundaries The ability of the VP ITC to collect quality data at a given concentration does not guarantee that the isotherm can be fit When a concentration is too high the isotherm becomes too steep C gt 3000 to obtain a unique fit When this occurs the data collected may represent the fitted K or any K of greater magnitude Also when concentrations are too low C lt 10 the fitting routines can have trouble converging on the data This happens because we have only collected data on the tail end of the isotherm There exists a range of concentrations over which an acceptable isotherm can be obtained This range of concentrations for any sample corresponds to a C value between 10 and 3000 or optimally between 50 and 500 The maximum and minimum concentration curves can be computed and plotted with the activation of a switch Simply select the Show Max Min Opt Curves in the Advanced Options box lt Keep C Constant fay Keep Concentrations Constant Show Max MinfOpt Curves The maximum and minimum concentration curves are then plotted green and light blue respectively along with the simulated experimental curve black The fourth curve is the optimal curve blue The optimal curve represents a C value of 100 By default the simulated curve will be the same as the optimal curve until the simulated curve is adjusted Simulation Macromolecule Conc 0 10 mM Maximum Macromolecule Conc
13. nce C has no meaning for the Two Site model the C control and the maximum minimum and optimal curves will not be available 0 lt a 2 Se fee SF 200 gi 400 U Oo T 60 amp so ee 800 4000 00 05 40 15 20 25 30 35 40 45 50 55 Molar Ratio 13 start New Session start Experimental Design V Vizarc Add New Curve Export to IMJ File Input Mew Bind Parameters Input New Run Parameters Maximize Ciuality Minimize Sample Print Simulation Plot Export to OH File Buttons Start New Session This button clears the simulation plot and allows the operator to start over in the simulation process Add New Curve This button saves the current simulated curve to a temporary plot A new simulation session will begin with the previous curve still plotted This is ideal for comparing two different simulations Input New Bind Parameters This button allows user to jump to new binding parameters without going through several iteration steps Input New Run Parameters This button allows user to jump to new run parameters without going through several iteration steps Print Simulation Plot The Print button will print the simulation plot Export to DH File This button exports a DH file The DH file can then be imported into ITC data analysis and fit 14 Switches Show Kd The Show Kd switch will allow users to work with and adjust Kd Ka is displayed by default Show
14. nema ayn mT ee CRT n eran CRON Tene mentee Ute etree enone E 14 DWC HES boils s i tr Aahidnck i aah ahs a Ae a Ih he iA alt tah ah LAU al Ai islet hh ls ies 15 SECO o Dame COSIO TIONS see T E ols Si are aoa eae ic vets eR OT 16 Sample Prepar on RECOMMENdAN ON Serei n N E A OTO 16 Sampe SOMMOn Pre paralOn seie N N E E T O ce ieleaas at eat le 16 Concentrating and Filtering Samples eeeeeeeeeeeeeereererererererrerrerrrerrrrerrrrrrrrrrrererrrrererererrerrrererrrrrrrrrrrrrrrrrrees 17 Sample Concentration Measurement csser E E esse A E AE I7 Temperature Dependence of Binding Heats ersciirernrisui rani A T E wicionce de races oeeeotes 17 Socnonm4 Troubleshoot srin i o a e r 18 Way is my Ionen too Seep arna a a a a N 18 Why does my Isothermnot look ikea Slamold nerenin ee EAE E E E E E A 18 Disclaimer Simulated data from the ITC Expert software is not a replacement for actual experimental runs on the VP ITC Section 1 ITC Expert Background ITC Expert will quickly and conveniently simulate an ITC binding experiment within the Origin 7 0 software application Based on a specific set of sample and experimental conditions ITC expert will automatically generate a simulated binding isotherm ITC Expert then allows the operator to change the sample and experimental conditions and the simulated binding isotherm will be appropriately updated to reflect those new conditions Finally the ITC Expert utility will allow the operator to save the d
15. nt 0 14 a aes 0 16 0 0 0 5 1 0 15 2 0 Molar Ratio Adjusting the Simulated Curve Once the simulation curve has been created every parameter can be adjusted The simulated curve will then be recalculated and plotted If the experimental parameters number of injections injection volume cell concentration or syringe concentration are adjusted the maximum minimum and optimal concentrations will remain constant If the binding parameters N K and DH are adjusted the maximum minimum and optimal concentrations will change accordingly To adjust the parameters simply click on the toggle arrows K and DH will increase and decrease by a half an order of magnitude of its current value N N Stoichiometry 1 qa K CIM iEp fa DH CalfMol 1 5Ep fal Cell Concentration m sok E f Wi Syringe Concentration mM a Ja J ranama AE eman ede paj 10 Adjusting the Simulation Out of Range If you adjust the simulation curve to a situation where the heats no longer meet the minimum requirement the simulation curve will turn red and a warning message will appear This situation is most likely caused by too few injections or too weak concentrations The requirements for the curve to be in range are a total heat of 50 uCal and a minimum heat of 5 wCal in the first injection AH KCal mole of Injectant 10 12 14 Warning Injection heats are too low a
16. pparent from looking at these isotherms that their shape is reasonably sensitive to binding constant only for C values in the range 10 lt C lt 3000 corresponding to binding of intermediate strength We will refer to this range as the experimental K window When available the middle of the window from C 50 to 500 is most ideal for measuring K The ITC Expert software utility will use C 100 as the ideal situation for experimental design See the graph below for some example C curves c 500 AH c 4 0 c 4 Kcal Mole Injectant c 1 0 N 0 0 i i Xtor concentration of injected solute in the cell before each injection Mror concentration of macromolecule in the cell before each injection after correction for volume displacement The x axis on the graph above is expressed in terms of Molar Ratio The correct choice of macromolecule concentration for an experiment depends upon the magnitude of K C Limitations High C When C is too large greater than 3000 the curvature in the binding isotherms for binding systems with varying Ks will no longer appear unique Below is an isotherm where K 10 and C 10 Notice how the curve is nearly a step function 0 es ERBRE BREE i P AH KCal mole of Injectant 10 HBHEEERHEeEeEHR Ee 6G 0 0 0 5 4 0 4 5 2 Molar Ratio The next curve is an isotherm where K 10 and C 10 C in this case is an order of magnitude greater than the previous isotherm howe
17. r example suppose that you are currently working at 30 C and have obtained a fitted value for DH 4 kcal mol Because binding enthalpy is temperature dependent every degree that you increase the temperature of an experiment will increase the binding enthalpy 0 3 to 2 kcal mole The raw heats will increase as well In this case if you increase your experimental temperature to 37 C then you should obtain a binding enthalpy between 6 4 and 18 kcal mole higher raw heats greater signal to noise and a better defined binding curve This is accomplished simply by changing the temperature of your experiment You do not need to change the concentrations of your reactants Alternatively you could conduct the experiment at a lower temperature Consider the above example If the same experiment were performed at 5 C then the binding enthalpy and raw heats will become more endothermic If the DCp 1s 1 kcal deg mol then reducing the experimental temperature to 5 degrees will yield a binding enthalpy of 21 kcal mol The raw injection heats will be endothermic as well and of greater magnitude than the original exothermic heats observed at 30 C Since the DCp is usually linear and negative then a measurement of binding enthalpy at two different temperatures will allow prediction of binding enthalpy at any temperature by fitting the data to a straight line Heat capacity change of binding is not a new concept Long time users of MicroCal instruments have
18. the larger the observed heat effect will be Therefore each reaction pair component pH should be checked prior to performing an experiment and adjusted if necessary 16 Concentrating and Filtering Samples A common process in sample preparation for any biological binding experiment is the concentrating of sample Samples are concentrated to obtain a suitable concentration for the binding reaction to be observed Detection limits of instrumentation are often high enough that protein sample must be concentrated to micro and nano molar levels 0 5 to 3 mg ml Care must be taken in concentrating protein samples since contaminants may be introduced into the sample solution resulting in solution mismatch or particles that may produce heat during stirring Spin columns are often used to concentrate protein samples If spin columns are going to be used then the following procedure is recommended First the samples should be dialyzed into the appropriate buffer Next the column should be washed typically with deionized water Be sure to adhere to the manufacturer specifications and protocol for column washing to ensure that the filter preservatives have been completely removed Once the column has been thoroughly washed the final dialysis buffer should be used to equilibrate the spin column see manufacturer guidelines Now the column is ready for concentrating the protein Following the concentrating process and before degassing the sample should
19. ver the curve has not significantly changed We could easily fit this isotherm to a model using Ka 10 10 or even greater values 0 BPHEERHEEEHEEESE NEE AH KCal mole of Injectant 10 HBHEHReEHEeEHREe 6G 0 0 0 5 1 0 1 5 70 Molar Ratio To enable the isotherm to be accurately and uniquely fit we need to keep the C at a value of 3000 or lower preferably at 500 or below Low C When C 1s too low the isotherms become too flat to be distinguishable Keeping the C value above 10 preferably 50 or higher will ensure that the resulting binding isotherm is not featureless and that it can be properly fit Concentrations Ratio The syringe concentration should be approximately 15 times the cell concentration The cell concentration 1s the critical concentration of the two and it will be used to determine the syringe concentration The cell concentration will define the C value but the syringe concentration along with injection volume will only determine the spacing of points on the x axis of the binding isotherm Because of this the concentration of the syringe can really be anywhere from 10 to 20 times the concentration of the cell concentration The ITC Expert simulator sets the syringe concentration at 15 times the cell concentration which is an appropriate choice for your experiments Section 2 ITC Expert Controls The Experimental Design Wizard Walkthrough The ITC experimental design wizard is an automated program th

Download Pdf Manuals

image

Related Search

Related Contents

  Etac Molift RgoSling  Installation, Op - comatec wesper airwell  G.F. 4 Jet Lift  INSTRUCTION MANUAL  Información del Documento Original  RG27203 - 極東製薬工業株式会社    baixar manual - ECB Ar Condicionado  Dual Laser InfraRed (IR) Thermometer  

Copyright © All rights reserved.
Failed to retrieve file