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KE User Manual - Metrohm Autolab

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1. Figure 3 9 Selecting a Region for the Average to be calculated d The Overlay s baseline should be now zero as shown in the figure below Click OK to close the Signal Processing dialog Signal Processi ng Action Zero at Average v falue The average value of the elements in the selected area will be set to zero This will effect the whole curve Apply To Full Plot All Plot 1 Plot 2 Plot 3 Plot 4 Plot 5 Selected Region Se x k Pointer yp Zoom E 1Select Legend Response m 1500 Time s Figure 3 10 The Corrected Baseline of the Overlay Autolab SPR KE User manual 21 3 4 Create a Project File In this step the Overlay file will be added to a new Project file a Click the New Project item in the Activity Bar as shown in the screenshot below New Overlay New Pray New Ber In New Vant Hoff Project Measurements 5 Overlays 1 Projects Calibrations Vanf Hoff Project Figure 3 11 Selecting the New Project item b Select your Overlay in the dropdown box in the Project Builder dialog as shown in Figure 3 12 Press OK to create the new Project Create New Project impot an opened Ova fie O Import an Overlay file from Disk Create New Vant Hoff Project Figure 3 12 Creating a new Project with the Project Builder c The new Project will be created and displayed in the KE application as shown in Figure 3 13 A copy of the O
2. Response m an co oOo a 18 07 28 07 38 07 48 07 5e 07 6e 07 78 07 8e 07 Time 5 Figure 7 12 Van t Hoff analysis requires four equilibrium analyses Including less than four will disable the Next button Step 2 of the Van t Hoff analysis is to do the actual fit Click on the Next button to show the second panel of the Van t Hoff Wizard This panel is shown in Figure 7 12 The results of Ka versus 1 T are plotted in the graph window The results for the fit to Equation 7 1 are shown in the summary area The results for AH AS and AC as evaluated at the reference temperature T can be copied to the clipboard for importing into other programs Autolab SPR KE User manual 123 Van t Hoff Analysis Wizard Step 2 of 2 max Legend Pointer 4 Zoom Van t Hoff Analysis Results R Pointer Ji Enthaply AHT 9537 3 317 9 cal M Entropy AS T cal M K w Heat Capacity 984 5 77 4 ACT I cal M K Copy To Clipboard D 2 pz 25 o a o N o 10 0 0032 0 00325 0 0033 0 00335 0 0034 0 00345 0 0035 0 00355 UT 1K Figure 7 12 The results of the Van t Hoff Analysis Once the analysis is complete the Van t Hoff Project can be saved 124 Autolab SPR KE User manual 8 Monte Carlo Wizard 8 1 Introduction Monte Carlo is a technique that uses pseudo random numbers to vary the fit This technique is used to cross che
3. Modeling Wizard Step 4 of 5 ly Start Rate Equation lel Model Information i ae k Define Species MB Equation Editor O Summary Defined Fate Equations Analyte Ligand Product A B AB Delete Figure 5 6 Completed rate equation 5 1 5 Summary of the Model The last step of the Modeling Wizard is the summary of the completed model To make changes to the model click the Back button to return to the step that requires changing Figure 5 7 shows the summary for the Monophasic model that was constructed in this example Modeling Wicard Step 5 of 5 be Start Model Information wl fee rer Name Flonophasic Model Standard 1 1 interaction model be Define Species be Equation Editor M Summary Defined Rate Equations Analyte Ligand A B Figure 5 7 Summary of the newly created model 58 Autolab SPR KE User manual To end the modeling wizard click the Finish button Before the wizard closes the user will be prompted to save the model 5 1 6 Editing an Existing Model To edit a previously saved model start the Modeling Wizard and select the Open Existing Model option in the first step of the wizard as shown in Figure 5 8 The selected model will be loaded into the wizard and all the model information will be automatically entered into the wizard Modeling Wizard Step 1 of 5 E Start O Model Information O Define Species Create New Kinetic Model OC Equation Editor Define
4. New Subcurve Measurements Overlays 5 Projects Calibrations Van f Hoff Project 1 E a no o a no a oe View Interaction Plot ew SPR Curves View Data Points View History Analysis Analysis Wizard alibration Wizard Onve rsion 100 Modeling Wizard Signal Processing Van t Hoff Wizard Monte Carlo Wizard 1500 Time s Autolab SPR KE User manual 119 Figure 7 8 The Van t Hoff Project in the main KE window after all the equilibrium analyses are complete The project navigator shows seven overlay files and seven analyses 120 Autolab SPR KE User manual 7 6 Van t Hoff Analysis Wizard Once the requisite equilibrium analyses are completed the user can launch the Van t Hoff Wizard by selecting Van t Hoff Wizard from the Activity bar This is shown in Figure 7 9 Analysis Wizard Modeling Wizard Signal Processing Van t Hoff meer ty Figure 7 9 Launching the Van t Hoff Wizard from the Activity bar Step 1 of the Van t Hoff Wizard is to select the Ka fits that will be used in the Van t Hoff Analysis Figure 7 10 shows the first panel of the Van t Hoff Wizard On the left is a table of the Equilibrium Analyses The first column of the table shows the temperature of measurement The temperature is the average of the temperature measurements in each of the measurement in the given overlay If the standard deviation of the temperature average 1s greater than th
5. User manual LCE ge T ESPRIT Kinetic Evaluation Version 5 3 Metrohm Autolab B V Copyright 2010 Table of Contents Nise TVET OCC OIA sinh P SE hace tte RENSES TEDE NERE ieee EEE SET ERE SERENE FORENE LEVE NSB SETE SEES SVENN l De WOtkin se With R E nania a E 2 2 1 User interlace OVETYIEN era senudwosadesevansinacaesnadciiinndenedevaadedesendiaacaesnanediinndswedeaadeddes 2 DD VA Men o E ee eee eee SEERE 3 ig De MY INU sara tat cet E E aera tA irae ace ec ae att ir E E A SET ONDE ES 3 EEEN TEEN eae een eee PE ce ne ge ee nnn te ee eee 5 23 2 TOOLS MENU or serene sne eet a sae eneret eee 7 Zi STAC WIN COU sa acicdee anced chic usaauannatace E nem civanausnaiateer 7 ZA VOOM AR ssi sperasist ats iahacecesteh R tes incomaasee 9 SEA EVIL BAR 5 ds re erne a 10 230 SP TOjeCt INAV LO atten Baf scn e a seeren 12 26 1 Editine N visation Bar ICM sacrer tiie ventana ty 13 2 6 2 Removing Navigation Bar Items cccccssseessseeeecccceneeesssseeeseceenees 13 203 Reana yzna A MALY SIS fact acti vawicaty n a teh cenaaw coat unteeualareeai eee 14 3 Getting Started with the KE Workflow ccccccsssesssseeeccccceneessseeececeeeneeeesses 15 a Opening Measurement Piles arisi EEE EER 15 512 Creatine AN OV EMIAY a T iew aa eae acne 16 3 3 Periorml Sinal Processin on 5 sr tea Nana niu twa wecea ees rad are rebel sends 18 J Cicate a Proet F Cosi A D 21 3 5 Using the Analysis Wizard to Fit the Da
6. Fie View Tools Help D g he p2 Zoom Hl Manage Overlays KIN101 1B0 KIN102 1B0 KIN103 1B0 KIN104 IBO KINT05 IB0 Overlay1 kod Project kpf History 21 06 2010 11 08 55 AM Added KIN101 Channel 1 Channel 1 from C Scimatic EcoChemie svn DA D ata KIN101 1BO 21 06 2010 11 08 55 AM Added KIN102 Channel 1 Channel 1 from C Scimatic E coChemie svn DA D ata KIN102 1BO New Calibration 21 06 2010 11 08 55 AM Added KIN103 Channel 1 Channel 1 from C Scimatic E coChemie svn DA D ata KIN103 1B0 New Van t Hoff Project 21 06 2010 11 08 55 AM Added KIN104 Channel 1 Channel 1 from C Scimatic E coChemie svyn DA D ata KIN104 1BO 21 06 2010 11 08 55 AM Added KIN105 Channel 1 Channel 1 from C Scimatic E coChemie svn DA D ata KIN105 1B0 21 06 2010 11 18 15 AM Signal Processing Zero at Average 5 21 applied to KIN101 Channel 1 KIN102 Channel 1 KIN103 Channel 1 KIN104 Channel Open Files A Measurements 5 Overlays 1 Projects 1 Calibrations Van f Hoff Project Views A View Interaction Plot View Data Points View History Conversion Tool Modeling Wizard Signal Processing i gt Copy to Clipboard Figure 4 12 History items of an Overlay file The history log keeps track of what files were used to construct the Overlay file and what if any actions were taken on the data with the Signal Processing Tool see section 5 2 It is p
7. KE User manual 131 The user can copy the results of the Monte Carlo runs to the clipboard These results can be pasted into a text editing program or a spreadsheet such as Microsoft Excel The summary will show the Chi Squared for each run of the fit and the parameter values A B C D E 1 Number of Iterations 100 2 3 Number of Plots 5 Variation 100 Fit ChiSq Ks E R 0 366 5493 0 001782 217 5192 4 56916 366 5493 0 001782 217 5192 4 56916 10 366 5493 0 001782 217 5192 4 56916 11 Son SO NAIR 717 5197 4 SAATA A a 6 KIN1O1 Differential T B g Figure 8 10 Copy to Clipboard results 132 Autolab SPR KE User manual Index bo 38 kcf 105 ksc 38 kvf 109 Spr 38 txt 38 A Activity Bar 2 9 Analysis Wizard 70 Analyte 53 Association 72 B Background Color 48 Biphasic 77 Blank Subtraction 58 B maxs 91 Border Color 48 C Calibration Builder 99 Wizard 100 Calibration analysis 99 Chart Properties 46 concentrations 73 Create Files 10 D Data file 38 Data Point View 33 Delete Selection 59 Depletion Correction 92 Determine Concentrations 72 93 Dissociation 72 E end time 75 Equilibrium 72 Equilibrium Analysis 87 Event Log 35 F Fast Fourier Transform 61 File Menu 3 G Global Analysis 81 H Help Menu 7 History 36 Horizontal Axis 47 Integrated Rate Equation 77 Interaction Plot View 32 K Kp 91 KE Sub Cur
8. The blue curve shows the fit using the Lorentz analysis The red curve is the standard Gaussian fit In the figure the two curves are nearly identical indicating that although the second data point might appear to be an outlier it 1s not Analysis Wizard Step 5 of 7 ole Legend Pointer j gt Zoom Set Plots to Exclude from Final Fit gt KIN101 Channel 1 KIN102 Channel 1 KIN103 Channel 1 KIN104 Channel 1 KIN105 Channel 1 18 07 28 07 38 07 48 07 5e 07 6e 07 7e 07 8e 07 Concentration M Figure 5 64 Outlier analysis showing the Lorentz fit blue and the standard Gaussian fit red The table on the left allows the users to manually exclude data points that the user believes are outliers When a data point is excluded the Lorentz curve blue remains the same while the Gaussian fit red 1s updated to ignore the excluded points Figure 5 65 shows the plot with two excluded points the red X data points Autolab SPR KE User manual 95 5 Analysis Wizard Step 5 of 7 oe Legend Pointer 4 Zoom Set Plots to Exclude from Final Fit KIN101 Channel 1 KIN102 Channel 1 KIN103 Channel 1 KIN104 Channel 1 KIN105 Channel 1 1e 07 2e 07 3e 07 4e 07 5e 07 6e 07 7e 07 8e 07 Concentration M Figure 5 65 Outlier analysis with two excluded data points 5 3 11 Equilibrium Analysis Analysis The last two steps of the Equilibrium Analysis do not require user interaction Once the con
9. 2 6 3 Reanalyzing an Analysis To reopen one of the analyses in a Project double click on the analysis name and it will be reloaded in the Analysis Wizard see section 5 3 Autolab SPR KE User manual 3 Getting Started with the KE Workflow 15 This section of the user manual gives an overview of a typical session using the KE application The following flowchart describes the workflow in this section d Open Measurement Files 4 Create an Overlay File sa Analysis Wizard to Signal Processing of Overlay fit the Interaction Plots Figure 3 1 Standard KE Workflow 3 1 Opening Measurement Files Create a Project File with the Overlay In this step we will be loading SPR measurement files into the KE application New Project New Calibration New Vant Hoff Project Measurements Overlays gm Projects Calibrations Van t Hoff Project Figure 3 2 Selecting the Measurements item a Click on the Measurements item in the Activity Bar as shown in the screenshot below 16 Autolab SPR KE User manual b The measurement files for this example are located in the Autolab SPR Data folder On the Open dialog change the Files of type to Data files ibo Select the following data files to open KIN101 ibo KIN102 ibo KIN103 ibo KIN104 ibo KIN105 ibo and click Open c The five files that were opened into the KE a
10. 3 2923E 10 Bmax 361 1407 0 7955 Concentration KIN102 gt Channel1 29900E 8 191 6143 KIN101 Channel 1 257 2020 KIN103 Channel 1 322 3266 KIN104 Channel 1 337 2287 KIN105 Channel 1 358 2322 Advanced Fit Details Copy to Clipboard 2e 07 3e 07 Concentration M Figure 6 10 Summary of a Calibration Analysis Figure 6 11 shows the Calibration project in the main KE window In the right panel is the project navigator here we have a Calibration file with the extension kcf the overlay from which the calibration was derived and the one calibration analysis under the node Calibration EET Fie View Tools Help D ca el EJ N Pointer 4 Zoom Legend pS Properties Calibrationt kef Calibration1 kcf Overlay1 kod Calibration Calibration New Project New Calibration New Van t Hoff Project Measurements Overlays Projects Calibrations 1 Van f Hoff Project View Interaction Plot View Data Points View History Calibration Wizard fodeling Wizard Signal Processing an t Hoff Wiz 5e 08 1e 07 1 5e 07 2e 07 25e 07 3e 07 35e 07 4e 07 45 e 07 5e 07 Concentration M Figure 6 11 A Calibration Project with one Calibration Analysis Autolab SPR KE User manual 111 112 Autolab SPR KE User manual 6 9 Using Calibration Files As discussed in section 5 3 2 one can use calibration files saved to a kcf file to set
11. B Monte Carlo Analysis Wizard Step 2 of 3 Run Monte Carlo Legend A Pointer Run 1 of 100 fo 0 8 0 6 Parameter Original Value MC Average MC s Ks 0 0018 0 0 E 215 8559 0 0 R 0 6 7520 0 0 127 JER Cancel Figure 8 5 Start of running the Monte Carlo Once the user clicks Start the Monte Carlo runs begin There is a progress bar that indicates the number of completed runs While the Monte Carlo is running all buttons except the Stop button are disabled As the runs progress the table of the Monte Carlo average and standard deviation are updated The histograms also update as a Monte Carlo run is completed This progress is Shown in Figure 8 6 128 Autolab SPR KE User manual Monte Carlo Analysis Wizard Step 2 of 3 Run Monte Carlo Legend Pointer Run 18 of 100 KINTOT Differential KIN102 Differential KINS T gt Parameter Original Value MC Average MCS Ks 0 0018 0 0017 0 000 E 214 0621 211 5725 23 74 R 0 9 0484 10 0499 9 552 8 997 1 042 Figure 8 6 The Monte Carlo wizard when the Monte Carlo is running Figure 8 7 shows the second panel of the Wizard when all runs of the Monte Carlo have finished A Gaussian plot with the average and standard deviation of the parameter values is overlaid on each histogram Monte Carlo Analysis Wizard Step 2 of 3 Run Monte Carlo Legend Pointer Run 100
12. Clipboard 3e 08 42 08 5e 08 6e 08 7e 08 9e 08 Concentration M Figure 5 55 Summary for the Integrated Rate Equation Fit Autolab SPR KE User manual 89 Summary Region Association Model Monophasic Model Method Numerncal Integration Advanced Settings Step Size 0 01 Number of Steps 10 lterations Per Step 1000 Start 135 Stop 900 Chi Squared 3 78S2E 4 Number of Iterations 9 Parameters 4 Data Points 766 Reduced x2 49 6480 Parameter Value Error b b 353 2540 02472 kal B S685E 4 53 3067 0 5703 kd 0000S S 7S57E 6 0 8191 Advanced Fit Details Copy to Clipboard Figure 5 56 Summary for the Numerical Analysis Fit Once fitting procedure has been completed it is possible to view the goodness of fit of the model using a Residual plot To view the Residual plot click Plots gt Residual from the Toolbar as shown in the figure below Legend k Pointer pa Zoom Plots Analysis Plot Residual Plot Both Flots Figure 5 57 Displaying the Residual Plot The Residual plot shows the difference between the model and experimental data A model that is a good fit will have a residual plot where all the data points are randomly scattered around zero Figure 5 58 is a screen shot of a Residual plot for the example data From this plot we can see that the model selected does not fit the data The next step would be to repeat the analysis process with the Biphasic model 90 Autolab
13. In this case the warning dialog shown in Figure 5 77 will appear Autolab SPR KE User manual 103 Warning i One or more concentrations could not be determined from this calibration curve as the intensity data point is greater than Bmax of the calibration curve C Don t show this dialog again Figure 5 77 Warning for invalid concentration calculation This inconsistency is shown in Figure 5 78 The Reguilibrium for KIN105 is 357 41 which exceeds the Bmax of the calibration curve which is roughly 340 In this case it is not possible to calculate a concentration for KIN105 and that entry is empty in the table pris Wizard Step 4 of 4 SF Enter the Plot Concentrations Legend Pointer g gt Zoom Use Calibration to Set Concentrations Open Calibration File C Documents and Settings Jim Use existing calibration i Req tJ Concent Unit Maximurr KIN101 mi gt Channel 286547 nM 250 08 KIN102 Lv Chamel 59713 mM Z844 I Req is greater than Bmax KIN103 Seay firs nm 32237 of calibration curve KIN104 m ag Channel 1 459 54 nM 336 68 Fa 357 41 v 1e 07 2e 07 3e 07 5e 07 6e 07 7e 07 8e 0 4e 07 Concentration M Figure 5 78 Missing concentration At this point the user should load a different calibration curve and execute a new concentration calculation 104 Autolab SPR KE User manual 6 Calibr
14. The Blank Subtraction action subtracts the selected plot from the other plots that are selected in the Apply To section of the Signal Processing dialog Blank Subtraction can execute on a selected region or the full plot To use the Blank Section action select the plot that you wish to use as the blank as shown in Figure 5 14 This plot will then be subtracted from the other selected plots Action Blank Subtraction w Flot H ka The selected plot will be subtracted from all the checked plots Figure 5 14 Blank Subtraction Options 62 Autolab SPR KE User manual The following screenshots show the effect of using the Blank Subtraction action The effect of the blank subtraction is can be seen in Figure 5 16 as the Response intensity has been reduced Legend k Pointer pa zoom r Select Response m s50 Time s Figure 5 15 Overlay before Blank Subtraction Legend k Pointer pa Zoom Select pe ko Response Mm 0 6 cm t s50 Time s Figure 5 16 Response Intensity is reduced after Blank Subtraction 5 2 2 Delete Selection The Delete Selection action deletes the selected region of the plot This action is available only for a selected region and cannot be used with the Full Plot option Autolab SPR KE User manual 63 The selected region of the plot is removed while any plot information before and after the deleted region is retained The data that remains
15. Title Overlay plot Vertical Axis Label Response rm Markings Arial 10 Horizontal amp xiz Label Time 2 Markings Anal 10 Figure 4 27 Chart Properties The last tab in the Chart Properties dialog see Figure 4 28 contains the configurable items that are global the same for all opened files The Border and Background colors can be changed while the Vertical and Horizontal gridlines can be shown or hidden Chart Properties Global Colour Scheme arid Lines Border Show Vertical Lines Background Show Horizontal Lines Figure 4 28 Global Properties 4 7 Printing Data In KE it is possible to print the graphs and the files details of the graphs 52 Autolab SPR KE User manual To print a file click the File gt Print menu item and Figure 4 29 will be displayed rint Job Builder Prin Select the data to be printed po hart Deae p Pas Marans F Overlay kod Dissociation om Left Fight enii E quilbrium Top Bottom Page Orientation Portrait Landscape Figure 4 29 Print Dialog From this dialog it is possible to configure the items that are to be printed Figure 4 29 depicts a project file with all of its graphs to be printed To print file details click the appropriate check box in the Details column The Page Margins and Page Orientation can be also configured from within this dialog It is also possible to preview a file
16. User manual 65 a FFT Frequency Response Legend k Pointer j Zoom 1 1 Select Plot 1 Magnitude m 0 04 0 05 0 06 Frequency Hz Figure 5 20 Region of the spectrum to retain After selecting the region to retain for each plot the filtered plots will be presented as shown in Figure 5 21 Legend k Pointer P Zoom rt Select o a T gt on T Ma 50 Time Figure 5 21 Result of the Fast Fourier Transform 5 2 4 Mathematical Operation 66 Autolab SPR KE User manual The Mathematical Operation action performs the operation that is selected by the user on the selected plot s The available operations as shown in the figure below are Addition Division Multiplication and Subtraction To use this action select the operation that you wish to perform and enter in the Value to assign to this action Next select the plots that this action will be applied to and select the region of the plot s that will be affected it is also possible to use the Full Plot option Action Mathematical Operation w Value 3 Subtraction Figure 5 22 Mathematical Operation Options Figure 5 23 and Figure 5 24 shows the result of multiplying one of the plots in the Overlay by three Legend k Pointer p Zoom r Select 1 un Cc a Z un 1 o 1500 Time Figure 5 23 Overlay before the Mathematical Operation is applied Autolab SPR KE User manual 67 Legend
17. Van t Hoff Temperature Van t Hoff Multiple overlays Section 7 based allowed analysis 4 6 Changing Chart Properties It is possible to change to look of the graph for Overlay and Project files By clicking View gt Chart Properties or the Properties button in the Toolbar will launch the Chart Properties dialog as shown in Figure 4 26 below Note It is not possible to change the plot properties of a Measurement file The KE application handles Measurement files as Read Only documents 50 Autolab SPR KE User manual 13 Channel 1 12 Channel EQUION Channel 1 Properties Pat se Clr Figure 4 26 Chart Properties Dialog The first tab in the Chart Properties dialog is Plots To change one of the plots in the graph click on its name from the list of plots Note It is possible to edit the properties of multiple plots at once Select multiple plots by holding down the SHIFT button while clicking on the plot names The Line Pattern Size and Color can be changed by using the dropdown lists on this dialog It is also possible to display Symbols on the data points in the plot by selecting a Symbol Pattern by default Symbol Pattern is set to none The next tab in the Chart Properties dialog is for the Chart itself As shown in Figure 4 27 the Title Vertical amp Horizontal Axis can be configured here Autolab SPR KE User manual 51 Chart Properties
18. by clicking File gt Print Preview in the main menu Figure 4 30 shows the print preview of an Overlay file Close 2010 06 21 01 29 PM 88 amp 8 8 8 Overlay1 kod Figure 4 30 Print Preview Dialog Autolab SPR KE User manual 53 5 Analyzing the Data This section of the manual provides an overview of the different features that are available in the KE application to analyze data 5 1 Modeling Wizard The Modeling Wizard is used to construct Rate Equations that are used by the Numerical Integration method in the Analysis Wizard see section 5 3 The process to construct a rate equation is performed in five steps The following subsections go over each step in detail 5 1 1 Starting the Modeling Wizard The Modeling Wizard can be opened from the Main Menu section 2 2 Tools gt Modeling Wizard or from the Activity Bar section 2 4 Modeling Wizard Upon opening the Modeling Wizard the dialog in Figure 5 1 will be displayed Modeling Wizard Step 1 of 5 E Start O Model Information O Define Species Create New Kinetic Model CO Equation Editar Define a custom Kinetic Model by defining the species and specifying the Rate Equation O Summary Open Existing Model kmd Open a Kinetic Model kmd that was previously saved Figure 5 1 Creating a model In Step 1 of the Modelling Wizard there are two options to choose from Create a new kinetic model or Ope
19. data on the Reg vs Concentration graph and the data points used to construct the graph The affinity constant Kp and the Bmax value are also presented The data on the summary step can be transferred to another application by clicking the Copy to Clipboard button 98 Autolab SPR KE User manual Gri Wizard Step 7 of 7 Joe Equilibrium Summary Legend Pointer Zoom Summary Region Equilibrium Affinity Constant KD 8 4041E 9 1 8047E 10 Bmax 294 4964 0 6314 Concentration Req Differential 2 5000E 8 226 4434 Diferertal 1 0000 254 4977 Diterental 2 00008 280 8020 Differential 4 0000E 7 290 9613 KIN105 Differential amp 0000E 7 302 1348 Advanced Fit Details Copy to Clipboard 1e 07 2e 07 3e 07 4e 07 5e 07 6e 07 78 07 8e 07 Concentration M Figure 5 68 Equilibrium Summary The summary data will be saved with the analysis and added to the Project once the Close button on the wizard is clicked The summary of the analysis will be still available by clicking on the Main Menu View gt Summary or by selecting the View Summary option in the Activity Bar 5 3 13 Determine Concentrations The analysis wizard can be used to set the concentrations of a set of measurements by applying a concentration Calibration curve Creating a Calibration curve is described in Section 6 To calculate the concentrations for a set of measurements from a Calibration select Determine Concentrations on the
20. dialog click the Signal Processing item in the Activity Bar as shown in the figure below Autolab SPR KE User manual 19 New Overlay New Project New Calibration New Van t Hoff Project New Subcurve Measurements 5 Overlays 1 Projects Calibrations Van t Hoff Project Modeling Wizard Signal Processi Van t Hoff Wizard Monte Carlo Wizard Figure 3 7 Selecting the Signal Processing item b The Signal Processing routine to remove the baseline offset is called Zero at Average Select this action from the list as shown in the figure below Action Blank Subtraction Delete Selection Fast Fourer Tranzform Mathematical Operation Normalize Figure 3 8 Selecting the Zero at Average Option c The Zero at Average will take the average of the selected area and subtract this amount from the entire plot Select a region in the flat part of the overlay using 20 Autolab SPR KE User manual the Selection tool and click the Apply button The screenshot below shows the selection of the area that will be averaged Signal Processi ng Action Zero at Average The average value of the elements in the selected area will be set to zero This willl effect the whole curve Apply To Full Plot All Plot 1 Plot 2 Plot 3 Plot 4 Plot 5 Selected Region es Legend Pointer j gt Zooh fF Select Tt N h oOo Response m tN 8 1500 Time s
21. gives an overview of the features of the Analysis Wizard A step by step example of how to use this wizard is given in the Getting Started with the KE Workflow section of the manual section 3 5 The Analysis Wizard can only be opened when a Project file section 4 5 is created or a saved Project file 1s opened from disk To launch the Analysis Wizard click Tools gt Analysis Wizard from the Main Menu or click on the Analysis Wizard item in the Activity Bar as shown in Figure 5 35 74 Autolab SPR KE User manual New Overlay New Project New Calibration New Van t Hoff Project New Subcurve Measurements 5 Overlays 1 Projects 1 Calibrations Vanf Hoff Project View Interaction Flot View SPR Curves View Data Points View History Analysis Wizard Calibration mela Conversion Tool Modeling Wizard Signal Processing Vant Hoff Wizard Monte Carlo Wizard Figure 5 35 Launching the Analysis Wizard from the Activity Bar The screenshot below Figure 5 36 depicts the first step that is shown when the Analysis Wizard is opened Autolab SPR KE User manual 75 faang Wizard Jog Select the Region to Analyze Legend Painter 4 Zoom 1 Association Phase in the interaction plot which the analyte combines with the immobilized ligand to form a complex Equilibrium Phase in the interaction plot where the observed rate of formation for the complex is zero M oOo Oo Dissociation Phase in
22. interaction plots you can click the Check All button Once the interaction plots have been selected press the OK button to create the new Overlay Figure 4 20 shows the results of the selections made in Figure 4 19 T For users of the Springle software the Channel 2 and Differential columns are not available Autolab SPR KE User manual 45 c Fie View Tools Help New Calibration New Van t Hoff Project YEW SUDCUIVE Measurements 5 Overlays 1 Projects Calibrations Van f Hoff Project 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 Time s Figure 4 20 The new overlay file The new Overlay file can be saved to disk by clicking File gt Save or click the save icon ld in the Toolbar In general any files that are opened in KE that contain unsaved data will appear with a at the end of their name 1 e the new Overlay name is Overlay kod 4 4 1 Overlay Manager Once an overlay is created it is possible to add or remove measurements from the overlay before it is saved Clicking on the Manage Overlay button on the Toolbar or Tools gt Manage Overlays to the launch the Manage Overlay Dialog 46 Autolab SPR KE User manual Th T ig agree ie area y anat L apja Select the plots to show in this overlay Available plots are indicated with a green background Overlay plot names can be edited by double clicking on the name Channel 1 Channel Differenti
23. k Painter P Zoom 15elect Response m 1500 Time 3 Figure 5 24 Multiplication of a Plot by Three 5 2 5 Normalize The Normalize action subtracts the offset of the first Response data point y axis from the rest of the plot This technique is useful to quickly remove an offset from the data This action is only available to the Full Plot Figure 5 25 and Figure 5 26 show the effect of executing the Normalize action Legend k Pointer j Zoom r Select 450 Response m F J a hl a o 1500 Time s Figure 5 25 Overlay before Normalize Action 68 Autolab SPR KE User manual Legend k Pointer Pr Zoom 15elect a a wn a ot wn a of 1500 Time Figure 5 26 Overlay after Normalize Action 5 2 6 Smooth The Smooth action performs a Savitzky Golay smoothing filter on the selected data This action can be executed on the full plot or the selected region within the plot s To perform the Smooth action select Smooth from the list of actions as shown in Figure 5 27 The polynomial used in the filter is set by the user either 2 or 4 order default 27 Order The number of points in the smoothing window is defined by the equation 2 m 1 where the value of m is determined by the user via the Value dropdown list see Figure 5 27 Action And Order ka 4 Savitzky Golay filter i used to smod 7 the plot with an n th order polynomial User does n
24. of all the species that will be used in the rate equations E Define Species O Equation Editor Name Name Delete hy B O Summary Figure 5 4 Defining the Species Define Species Only Once If the rate equation for the model is made up of multiple equations for example g SER em esi a L S QD i i i 56 Autolab SPR KE User manual 5 1 4 Creating the Rate Equations In step 4 of the Modeling Wizard it is time to construct the rate equation Use the dropdown controls on the dialog as shown in Figure 5 5 to configure the species for the rate equation Modeling Wizard Step 4 of 5 k Start Rate Equation Analyte Ligand Product TELLE o MEL de Add be Model Information be Define Species M Equation Editor O Summary Defined Rate Equations Analyte Ligand Product Delete Figure 5 5 Creating a rate equation Once the Analyte Ligand and Product have been defined click the Add button to add the rate equation to the model A valid rate equation must consist of at least an Analyte and a Product Figure 5 6 shows that the model has one rate equation defined for it If you wish to remove a rate equation from the model then select the equation from the defined equations click on it to highlight it and press the Delete button Once you have configured your rate equation click Next to proceed to the next step Autolab SPR KE User manual 57
25. only Overlays that are a separate file kod or part of a Project file kpf can be used in the Signal Processing tool The Signal Processing dialog can either be opened by clicking from the Main Menu Tools gt Signal Processing or clicking on the Signal Processing item in the Activity Bar as shown in Figure 5 11 60 Autolab SPR KE User manual Analysis Wizard Modeling Wizard Signal fons Figure 5 11 Selecting Signal Processing Once the Signal Processing dialog has been opened it will appear as shown in Figure Sel Signal Processing 7 ok x gt meg Reon Legend R Pointer y Zoom Select Baseline Correction fal 450 1 _ 400 Linear regression will be applied on the elements of the selected region The whole curve will be corrected for the 350 found slope the baseline drift J 300 Apply To Full Plot Selected Region All Plot 1 Plat 2 Plat 3 2 Plot 4 Plat 5 tN n o Response m N a o o h o Undo Apply 500 4000 4500 2000 3 Time s 2500 Figure 5 12 Signal Processing Dialog The following descriptions correspond to the numbers that appear in Figure 5 12 1 Actions Select which signal processing technique to perform on the data The following subsections will explain each of the available actions 2 Apply To This section is used to specify which interaction plots are to be included in the signal process
26. the Activity Bar Autolab SPR KE User manual 91 5 3 8 Equilibrium Analysis Equilibrium analysis uses a number of different steps from either Association or Dissociation analysis This section is for Equilibrium Analysis Only 5 3 9 Equilibrium Analysis Plots at Equilibrium The first distinction follows immediately after setting the start and end points for the plots In step 4 each plot is fitted to see if it has reached equilibrium First a dialog discussing the fit appears as shown in Figure 5 60 Info i Click the Fit button to fit the equilibrium curves The fit will determine which curves are with 1 of their fitted maximum value You can change the threshold value for considering fits at equilibrium in the textbox You can choose to exclude curves by using the checkboxes after the fit is finished C Dont show this dialog again Figure 5 60 Dialog indicating a fit for equilibrium will occur Once the dialog is dismissed the user has the option to test each plot to determine if it is at equilibrium The data table first shows the maximum value of Req determined from the data The maximum value as estimated from a fit is not yet shown To fit the data click on the Fit button This is shown in Figure 5 61 Each curve will be considered at equilibrium if e Its maximum value from the data and from the fit are within the threshold value in Figure 5 61 that is 1 e Or the maxi
27. the OK button Select the plots to add to the new overlay Available plots are indicated with a green background weray plot names can be edited by double clicking on the name dr nl KIN101 KIN 102 KIN 103 KIN104 KIN105 Figure 3 5 Selecting the Plots for the New Overlay c A new overlay file will be created and added Notice in the screenshot below that there is now a 1 beside the Overlays item in the Activity Bar 18 Autolab SPR KE User manual Kinetic Evaluation 2 Oo E Fie view Tools Help DREK Zoom Legend E amp Properties j Manage Overlays KIN101 1B0 KIN102JBO KIN1O3 IBO KIN104 IB0 KIN105 JBO Overlay1 kod Create Files New Overlay New Project New Calibration New Van t Hoff Project Measurements 5 Overlays 1 Projects Calibrations Vanf Hoff Project hm on Views A a no c o a no vw N View Interaction Plot ew SPR Curves View Data Points View History Analysis _alibration Wizard Conversion Tool Modeling Wizard Signal Processing sek iel i za 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 arlo Wizard Time s Figure 3 6 The New Overlay d You can save this overlay to disk by clicking the Save Toolbar icon mj 3 3 Perform Signal Processing The overlay created contains an offset This can be removed by using the Signal Process feature in KE a To open the Signal Processing
28. the concentrations in the overlay by applying 4 calibration file Figure 5 38 Select the Region to analyze Autolab SPR KE User manual T1 Note Equilibrium Analysis contains different steps in the Analysis Wizard The Equilibrium Analysis process is described in the following sections a 5 3 8 5 3 2 Specify the Concentrations The second step of the Analysis Wizard is to specify the concentrations of the interaction plots As shown in Figure 5 39 the concentration value is entered and the unit of the concentration is specified 78 Autolab SPR KE User manual Enter the Plot Concentrations Concentration Urit KIN101 Differential 22 KIM 102 Differential aU KIN103 Differential 60 EJN104 Differential KINTOB Differential Figure 5 39 Specifying the Concentrations It is possible that the concentrations were already supplied before running the Analysis Wizard One such method to determine the concentrations is to run the Analysis Wizard and select Determine Concentrations from a Calibration curve This process is detailed in section 5 3 13 If the concentrations were determined from a Calibration curve the user will get a warning Warning Equilibrium Analysis Using Calibration gk You executing an Equilibrium Analysis where the concentrations were set from an equilibrium calibration curve This analysis using calibration curves derived fram Equilibr
29. vou wish to change and Use the line tool ta set the new value with the mouse Don t show this dialog again Figure 5 41 Instructions for Selecting Start End Times This dialog Figure 5 41 explains that there are two methods of setting the analysis times If you don t require this message it can be removed from the wizard by clicking the Don t show this dialog again checkbox and clicking OK The first method is to manually enter the times To enter a value double click the desired cell and enter in the time Figure 5 42 shows the end time for KIN101 Channel I being manually entered There are two Region Options to select on the dialog The default Same Region for All Plots will copy your entered time to all interaction plots The Unique Regions for Each Plot will allow the user to enter custom start end times for each plot 80 Autolab SPR KE User manual Set the Region to Analyze p KIM 101 Channel 1 KIN TO Channel 1 KIN TOS Channel 1 KIM 1 O4 Channel 1 KIM 105 Channel 1 Figure 5 42 Changing the End Point Manually The second method to change the analysis times is to use the graph s Line Tool to visually set the times The steps to use this method are Click single click on the time that you wish to change Click on the graph Line Tool Line button in the toolbar to activate it Drag the mouse pointer over the graph to select the des
30. 1 0 7612 0 7994 Figure 4 10 Overlay data copied in a Microsoft Excel worksheet 4 2 3 Event Log History Summary View The different file types in Kinetic Evaluation contain different details that can be displayed Event Log Measurement Files The Event Log in Measurement files is generated during the collection of data the Autolab SPR Data Acquisition software This data is read only in the KE application If the measurement file does not contain Event Log information then the grid will be empty The following screenshot shows the Event Log for a measurement 38 Autolab SPR KE User manual New Overlay New Project New Calibration New Van t Hoff Project New Subcurve Open Files A Measurements 5 Overlays 1 Projects 1 Calibrations Van f Hoff Project dre us E HEE 1191 3 eo Baseline 100 ul S 1071 4 ew 1071 4 ha 12 Baseline 100ul S 1188 3 11883 ch 1 1181 0 1181 0 ch 1 1177 2 1177 2 Ch 1 10375 1037 5 Ch 2 1171 3 2o a Ch 1 1026 Ch 2 1167 2 23 00 as O e Ch 1 Figure 4 11 Measurement Event Log History Overlay Files Overlay files keep an internal history of the changes made to the file These logged items can be displayed by viewing the History of the overlay file as shown in the figure below Autolab SPR KE User manual 39 Kinetic Evaluation Joey
31. 2 Equilibrium via extrapolation 5 3 10 Equilibrium Analysis Outlier Analysis Step 5 of an Equilibrium analysis will attempt to identify outlier points on the equilibrium plot Outliers are identified by using a Lorentz fit as described in the paper by Motulsky and Brown Standard fitting techniques assume that the data points are Gaussian distributed around the true values The Gaussian distribution has large tails and the fits are pulled by these tails to towards outliers The Lorentz fitting technique replaces the assumption that the data points are Gaussian distributed with the assumption that the data points are Lorentz distributed Because the Lorentz distribution has narrower tails it is not affected by outliers to the degree that the Gaussian fit is This technique allows for more robust identification of Outliers On entry into Step 5 a dialog box is shown that indicates if the Lorentz fit identified any outliers This dialog is shown in Figure 5 63 Motulsky and Brown Detecting outliers when fitting data with nonlinear regression a new method based on robust nonlinear regression and the false discovery rate BMC Bioinformatics 7 123 2006 94 Autolab SPR KE User manual Outlier Analysis l The Lorentz fit indicates no outliers Dont show this dialog again Figure 5 63 Dialog box indicating the presence or absence of outliers Figure 5 64 shows the results of the outlier analysis
32. 5 3 1 Selecting the Region to Analyze The first step in the Analysis Wizard is to specify which type of analysis is to be preformed Figure 5 38 shows the first step of the wizard The available analysis options are e Association Region in the Overlay where the analyte injected sample combines with the immobilized ligand sample on the gold disk to form a complex This is the region in the graph that has a positive slope e Equilibrium Region in the Overlay when there are no more ligand locations remaining for the analyte bind with This is the region in the graph where the slope of the line approaches zero e Dissociation Region in the Overlay where the analyte is removed from the immobilized ligand This region of the graph has a negative slope e Determine Concentrations The concentrations of an overlay can be set from a previously calculated Calibration curve See Section 6 for creating Calibration curves Section 5 3 13 details how to apply a calibration curve to an overlay to determine the concentrations Select the Region to Analyze Association hase in the interaction plot which the analyte ombines with the immobilized ligand to form a complex Equilibrium Phase in the interaction plot where the observed rate of formation for the complex is zero Dissociation Phase in the interaction plot where the analyte is removed from the immobilized ligand Determine Concentrations Determine
33. 61 0 9293 mi O 0 4823 0 6638 0 6908 Measurements 5 0 3416 0 4858 0 5176 Over i 0 2419 0 3321 0 3853 Projects 1 _ re ee 0 1694 0 213 0 321 Van f Hoff Project 0 1037 0 119 0 236 0 0327 0 0003 0 1052 0 0138 0 0653 0 035 T 0 0681 0 1402 0 1466 Ve 0 1168 0 2068 0 2054 Views A ew SPR Curves 0 1638 0 2233 0 2908 View History 0 2121 0 2697 0 3745 Ene 0 2683 0 3488 0 4323 0 3095 0 4057 0 5044 View Data Points L 0 3678 0 4952 0 5348 0 4227 0 5506 0 6612 0 4723 0 5848 0 7139 0 5175 0 6155 0 7543 0 5451 0 6753 0 795 06183 0 7455 0 8773 T T Figure 4 8 Viewing the data points of an overlay file For users of the Autolab SPR Springle software the Channel 2 and Differential plots are not available 36 Autolab SPR KE User manual It is possible to copy this data to another application via the clipboard To copy the data follow the steps below a Click on the columns that you wish to copy or click the square in the top left corner to select all the data as shown in Figure 4 9 File View Tools Help New Overlay New Project New Calibration New Vant Hoff Project New Subcurve Figure 4 9 Selecting all the data points b Press CTRL C on the keyboard to copy the data c Open the application that you wish to copy the data to Micro
34. Analysis method is similar to the previous process except the user can also load a custom model from disk The models in the drop down list as shown in Figure 5 48 are the models that are located in the Autolab SPR Models folder Autolab SPR Models When a model is selected the model is displayed and the description of the model is shown 84 Autolab SPR KE User manual Select the Model to Use Monophasic kroid s Selected Model Monophasic Model kiy Description Standard 1 1 interaction model Figure 5 48 Selecting the Numerical Analysis Method To select a custom model use the Browse button to select a Kinetic Model Description file kmd If the selected model does not already exist in the standard modelling folder then a prompt will be given to copy the selected model Figure 5 49 shows this user prompt Copy Selected Model Es 1 The selected model is not in the default model folder a Do you wish bo copy it there Figure 5 49 Prompt to Copy a Model to the Default Model Folder 5 3 6 Configuring and Fitting the Model The next step in the Analysis Wizard is the configuration of the fitting parameters On first open of this step the information dialog shown below will be shown to explain that for best results use the Test button to ensure that the initial parameters are in range of the experimental data In range means the model plots should be in the same order of magnitude as the experim
35. Calibration Wizard Modeling Wizard 3 gnal Process ng i 600 800 1000 1200 Monte Carlo Wizard a Figure 3 25 Analysis has been added to the Project This concludes the KE workflow demonstration The remaining sections in the manual will go into more detail about everything that was performed in this example Autolab SPR KE User manual 31 4 Working with Data This section of the manual provides an overview of the different file types that the KE application supports 4 1 Opening Files There are three methods to open file in KE The following subsections give an example of each 4 1 1 Opening a File from the Main Menu From the Main Menu Measurement Overlay Project and Calibration files can be opened Figure 4 1 shows the menu items Kinetic Evaluation Measurement Overlay Project induding Van t Hoff Calibration Figure 4 1 Opening a Measurement file from the Main Menu 4 1 2 Opening a File from the Toolbar Files can be opened from the Toolbar by clicking the open icon as shown in Figure 4 2 Kinetic Evaluation File View Tools Help er d Zoom E Legend f Properties Figure 4 2 Opening a File from the Toolbar 32 Autolab SPR KE User manual Different types of files can be opened Measurement Overlay Project and Calibration files Upon clicking the open icon in the Toolbar a selection for what type of file to open will be made as shown i
36. Clipboard button Autolab SPR KE User manual 29 Summary Region Association A Model Monophasic E Method Integrated Aate Equation UII Monophasic Kinetic Parameters ka 8723 7 1072 4 kd 0 002 0 0004 Advanced Settings Step Size 0 01 KINTOT Differential KIN102 Differential KIN 4 Concentration 25n Stark 135 47 Stop 900 Chi Squared 170 6580 Number of Iterations 27 Parameters 3 Data Points 154 Reduced x 2 1 1315 Parameter Value Error ies ks 0020 1 8504E 5 E 206 6979 0 8208 0 8987 R 0 2 2078 0 2795 O fee E A 0 208 9057 0 8671 Advanced Fit Details Copy to Clipboard Figure 3 24 Summary of the Fit 1 The Analysis Wizard is now complete Press the Finish button to add this analysis to the project j The analysis will be added to the Project and will be displayed in the user interface Notice that in the Project navigation bar see left side of the figure below the Analysis has been added To display the Overlay plot again right click on the Overlay name 30 Autolab SPR KE User manual File View Tools Help Project1 kpf Overlay kod New Project New Calibration New Van t Hoff Project psy SR eases YEW SUDCUIVE Measurements 5 Overlays 1 Projects 1 Calibrations Van f Hoff Project N am Response m View Interaction Plot n DO ew SPR Curves View Data Points View Summary Analysis Wizard
37. Curves View History Figure 4 6 Changing the View Mode via the Activity Bar The following subsections give an overview of each of the views 4 2 1 Interaction Plot View The Interaction Plot View 1s the data plotted in a graph This view is the default view for all file types Measurement files have the option to show hide the various plots that they contain This functionality is available via the Main Menu and is shown in Figure 4 7 By default all components of a Measurement file are displayed Channel 1 Channel 2 and Temperature or Differential data on the second Y Axis Autolab SPR KE User manual 35 Help Chart Properties Interaction Plots Channel 1 Data Points Channel 2 Event Log Temperature Differential Toolbar Activity Bar w Options Figure 4 7 Setting the visibility of the Measurement Plots 4 2 2 Data Point View The Data Point View displays the data points of the plots in a grid Figure 4 8 shows the Data Point View of an Overlay file File View Tools Help Zoom F Manage Overlays KIN101 1B0 KIN102 1B0 KIN103 1B0 KIN104 1B0 KIN105 1B0 Overlay1 kod O Time Plot KIN101 Time2 Plot2 KIN102 Time3 Plot3 KIN103 New Overlay b j5 2 6638 3 025 2 4219 New Project zi 1 7501 2 7078 0 5298 rare 017546 21033 2 0726 ME Frey 0 9589 1 5822 1 4976 0 7035 1 1941 1 1445 0 5683 0 88
38. REESE DES SEES ESS EEEED iors 49 4 0 Chaneime Chart Proper S 5 a rn ede ea date eder sas 49 Fe PARIS DA a Eee a a 51 SLANI ne e ata ea Ma caouen cae ai SES EGERN RESTER ERE dais 53 J d Modelno Wizi ee ae R ere 53 Sold Starting the Modeline Wizard scener telat itvertaeideed tons 53 5 1 2 Configuring the Modeling Information ccceecccsccececceeeseeeeeeeeeeeeeees 53 IE o DEMING Ne SPC CIOS eani n a RENEE raceme 55 JA Autolab SPR KE User manual 5 1 4 Creating the Rate Equations ccc ccessssscccccccseeessseeecsccceseeeesseseeseeeeunes 56 5 1 3 Summary olhe Model iirrainn nan erne 57 5 1 6 Editing an Existing Model cccccesssssecccccceeeeeeeeeeceeeeeeeaeeeseeeceeeeeeaaas 58 IAF OY ANCE SEES os parton taecetn sc suossenatwoesd E rer eres eler 58 Se SINA MD og 6X61 echt 1 on eee eee 59 2h I LAIN SO AC NO HEE EET A 61 D2 2 IC e SC ICC HOM shraatece E N E A EE 62 5 2 3 Fast Fourier Transform FFT 0 ce ecccsecccecccsecceescesseesssenseeeeseeess 64 5 2 4 Mathematical Operation cccccccccssssesseeccccceceeeeesseeecceeeeaeeeseeeeeeeeeeaaas 65 SE SENER O e ee eee eee eee ee ee 67 Dye 0 OO Ne da eet ENDE RESEN REFERERE ETERN ERE REDET 68 Dd VIC ROM e N I A E E E 69 SB ATO at AVTI Ger E E er reen 71 r IMAL SIS We dae EO E Een rese 73 5 3 1 Selecting the Region to Analyze sessssseeeresssssssseeresssssssseceressssssseeerees 76 5 3 2 Specify the Concen
39. SPR KE User manual Analysis Wizard Step 6 of 6 Jo Summary Legend RK Pointer Zoom Region Association Model Monophasic Method Integrated Rate Equation Advanced Settings Step Size 0 01 Number of Steps 10 Iterations Per Step 1000 Plot 1 Plot 2 Plot 3 Plot 4 Plot 5 Concentration 25nM Start 155 Stop 1630 Chi Squared 369 3120 Number of Iterations 12 Parameter Value Error Ks 0 0018 6 4995E 6 E 212 3246 0 2459 R 0 105 0783 0 2250 E R 0 317 4029 0 3333 m po Mt gt Response mm Copy to Clipboard 800 Time s Figure 5 58 Residual Plot The following figure is of an analysis in which the model fits the experimental data well Notice that the residual plot in randomly scattered around zero Analysis Wizard Step 7 of 8 od Legend OK Pointer P Zoom Plots Configure and Fit the Model 58 07 p 5e 07 Plot 1 Plot 2 Plot 3 Include this plot 4e 07 ChiSquared 4 4137E 12 Number Iterations 10 3e 07 Parameter Value i 28 07 Ks 120001 l i E 0 5000 4 0432E 8 18 07 Response m 2 5 Time s Figure 5 59 Example of a Well Fitting Model The summary data will be saved with the analysis and added to the Project once the Close button on the wizard is clicked The summary of the analysis will be still available by clicking on the Main Menu View gt Summary or by selecting the View Summary option in
40. a custom Kinetic Model by defining the species and specifying the Aate Equation O Summary Open Existing Model kmd Open a Kinetic Model kmd that was previously saved Figure 5 8 Opening a Model from Disk 5 1 7 Advanced Settings Certain models can have more than one product that contributes to the SPR signal For example in the set of equations that describe a conformal change reaction Ei Ez l A th px A BE AB AB both AB and AB can contribute to the SPR signal To indicate to the model that both AB and AB are final products click on the Advanced button in step 4 as shown in Figure 5 9 Autolab SPR KE User manual 59 Modeling Wizard Step 4 of 5 be Start Rate Equation Analyte be Model Information be Define Species MB Equation Editor O Summary Defined Rate Equations Analyte Ligand Product amp z AB AB hone AB Delete Figure 5 9 Advanced model configuration Clicking the Advanced button will show an additional dialog where the signal weights can be entered In Figure 5 10 the signal weights for AB and AB are set to be equal so that each of AB and AB contribute the same to the SPR signal in the model Advanced Settings Figure 5 10 Setting the signal weights 5 2 Signal Processing It is possible to improve data quality in the KE application by using the Signal Processing tool Signal processing can be performed on Overlay files
41. after the deleted region is moved to the end of the plot that is still remaining before the deleted region The following two figures are before and after screenshots of the Delete Section action Legend k Pointer 200m r 1 Select o a uw Z a Z uw a a 1500 Time Figure 5 17 Select a Region to be Deleted Legend k Painter j Zoom K Select ba oO Oo CH mm a a wn mm a 2 wn a of oO Lm 1500 Time Figure 5 18 Result of the Delete Selection Action 64 Autolab SPR KE User manual 5 2 3 Fast Fourier Transform FFT The Fast Fourier Transform FFT action is a filtering technique used to reduce high frequency interference in the plots This action can be applied to a selected region or to the full plot s The Figure 5 19 shows plots that contain high frequency noise Legend k Painter P zoom r 1 Select o a wn c a Z uw a o 50 Time s Figure 5 19 Noisy Data To improve the quality of the plots select the Fast Fourier Transform action and click the Apply button There is an intermediate step in the FFT action The user must select the region of the frequency plot they wish to retain for each plot that is being filtered In the frequency plot the data at the lower frequency is what is to be retained Select the area of the graph that contains intensity above the baseline Figure 5 20 depicts this selection Autolab SPR KE
42. al KIN101 KIN102 KIN103 KINT KIN105 Figure 4 21 Overlay Manager dialog before the Overlay is saved The check boxes will allow the user to add or remove measurements from the overlay Note ge plot names in the Overlay Manager by Double re Once the overlay is saved each measurement is saved as a single element in the overlay Figure 4 22 shows the Manage Overlay dialog after loading and overlay from disk Select the plots to show in this overlay Available plots are indicated with a green background Overlay plot names can be edited by double clicking on the name KIN10 Channel 1 KIN102 Channel 1 OO KINIO3Channel 1 KIN 104 Channel 1 KIN105 Channel 1 Figure 4 22 Overlay Manger dialog after the Overlay is saved Autolab SPR KE User manual 47 4 5 Project Files Project files are a new concept since KE version 5 0 The purpose of a project file is to store all analyses that were performed on an Overlay into one file 4 5 1 Creating a Project File To create a new Project file click File gt New gt Project or click on New Project in the Activity Bar The following dialog Figure 4 21 will be shown to assist the user in selecting how to create the new overlay Create New Project Import an opened Overlay file Oveday1 kod al Import an Overlay file from Disk Create New Vant Hoff Proje
43. apolation for a Calibration project 6 6 Outlier Analysis The calibration analysis looks for outliers in exactly the same way that the Equilibrium analysis looks for outliers using the Lorentz fit The outlier analysis is shown in Figure 6 8 Calibration Wizard Step 4 of 6 X Set Plots to Exclude from Final Fit Legend Pointer j gt Zoom Exclude KIN102 Channel 1 F KIN101 Channel 1 KIN103 Channel 1 KIN104 Channel 1 KIN105 Channel 1 2e 07 3e 07 Concentration M Figure 6 8 Outlier analysis for a Calibration project Autolab SPR KE User manual 109 6 7 Equilibrium Fit Step 4 is the Equilibrium fit for the Calibration project Figure 6 9 shows the fit to the data Catibration Wizard Step 3 of BAX Equilibrium Analysis Legend Pointer 4 Zoom Bmax 361 1 0 8 Maximum capacity of the prepared sensor surface Depletion Correction C Use Depletion Correction S R A R A E T Change Device Settings 2e 07 3e 07 Concentration M Figure 6 9 The Equilibrium analysis for a Calibration 6 8 Summary Figure 6 10 shows the summary for the Calibration analysis with the fit values for Bmax and Kp These values can be extracted to the clipboard if necessary 110 Autolab SPR KE User manual Calibration Wizard Step 6 of 6 ole Equilibrium Summary Legend Pointer 4 Zoom Summary Region Equilibrium Affinity Constant KD 2 6218E 8
44. ata Instead KE uses the fully integrated equilibrium equation 1 Req zg Ac Rmax P Kp TV A0 Rmax P Kp 4 Ao Rmax B S p a M V See for example de Mol N J and Fischer M J E Kinetic and Thermodynamic Analysis of Ligand Receptor Interactions SPR Applications in Drug Development Handbook of Surface Plasmon Resonance Ed Richard B M Schasfoort and Anna J Tudos Cambridge RCS Publishing 2008 123 172 f See equation 23 of Edwards P R et al Second Order Kinetic Analysis of Asys Biosensor Data Its Use and Applicability Anal Biochem 263 1 12 1998 Autolab SPR KE User manual 97 kuaa Wizard Step 6 of 7 ER Equilibrium Analysis Pointer 4 Zoom Req 5 am Bmax ig d Kn 6 1 40 1 E 9 Bmax 291 9 0 6 Maximum capacity of the prepared sensor surface Depletion Correction Use Depletion Correction en A R Al MV Analyte Mol Weight Ma 39000 DA Analyte Volume V 60 uL Device Surface Area S Ae mm Calibration Constant a 122g mm Change Device Settings 18 07 2e 07 3e 07 4e 07 5e 07 6e 07 7e 07 8e 07 Concentration M Figure 5 67 Equilibrium analysis after applying a depletion correction 5 3 12 Equilibrium Summary The last step in the Equilibrium Analysis is the summary This section is for Equilibrium Analysis Only The Equilibrium Analysis summary step as shown in the figure below shows the fitted
45. ation Analysis Calibration analysis is a special kind of equilibrium analysis It is used to derive a calibration curve that can be applied to Association or Dissociation measurements to determine the concentration values 6 1 New Calibrations To begin a calibration select New Calibration either from the activity bar as shown in Figure 6 1 or from the File menu This will start the process to create a new calibration project New Overlay New Project New Calibratior New Van t Hoff ject New Subcurve Measurements Overlays 1 Projects Calibrations Van t Hoff Project Figure 6 1 Creating a calibration project Once the New Calibration is selected the Calibration Builder dialog box is shown The Calibration builder is shown in Figure 6 2 Just like in the Project builder the user can select to add either an opened overlay file or to import an overlay from a file on disk Autolab SPR KE User manual 105 Create New Calibration import an opened Overtay fie Ovelayikod Import an Overlay file from Disk Figure 6 2 The Calibration Builder dialog 6 2 The Calibration Wizard Once the Calibration Builder is completed a new calibration project is created This project is shown in Figure 6 3 and it is very similar to a default project However only Signal Processing and the Calibration Wizard are available for calibration projects Kinetic Evaluatio
46. ations Legend Ik Pointer 4 Zoom Use Calibration to Set Concentrations Open Calibration File Use existing calibration Execute a Req Concent Unit eure KIN101 Differential v 228 76 Response m KIN102 v Differential 254 44 KIN103 v Differential 280 8 KIN104 v Differential 290 96 KIN105 v Differential 302 13 1500 2000 2500 3000 Time s Figure 5 73 Applying the Calibration curve Initially the user must select a calibration to use to calculate the concentrations The user can select to use e A Calibration file kcf loaded from disk as shown in the circled Browse button e A previously loaded Calibration which will be shown in the Use existing calibration combo box Once a calibration is selected the calculation can be executed as shown in the circled Execute button in Figure 5 74 FF FFF Analysis Wizard Step 4 of 4 BAX Enter the Plot Concentrations Legend Pointer Zoom Use Calibration to Set Concentrations G Open Calibration File C Documents and SettingsyJim Use existing calibration i a Concent Unit Maximum KIN101 v Differential 228 76 Response m vv kius 254 44 KI Differential KIN103 v Differential 280 8 KIN104 v Differential 290 96 KIN105 v Differential 302 13 1500 2000 2500 3000 Time s Figure 5 74 Loading a Calibration File in preparation to execute the calibration O
47. centrations and start end times are configured a Response at Equilibrium Reg vs Concentration graph will be plotted By nonlinear fitting of the data in the Reg vs Concentration graph as shown in Figure 5 66 the affinity constant Kp and Bmax values can be determined 96 Autolab SPR KE User manual TEER Wizard Step 6 of 7 cog Equilibrium Analysis Legend Pointer 4 Zoom A Req FA Bmax kd Kp Sra 8 4 0 2 E 9 Bmax 294 5 0 6 Maximum capacity of the prepared sensor surface Depletion Correction C Use Depletion Correction S R A R Alo 2749 V Change Device Settings 1e 07 2e 07 3e 07 4e 07 5e 07 6e 07 7e 07 8e 07 Concentration M Figure 5 66 Equilibrium Analysis The user can modify the results of the fit by making a depletion correction The depletion correction depends on the analyte molecular weight and the volume of the analyte in the Autolab SPR By default the analyte volume is 60 ul for the Autolab SPR The effect of the depletion correction is shown in Figure 5 67 Here a depletion correction was applied that changed both Kp and Bmax The standard way to apply a depletion correction is to first adjust all of the concentrations according to the formula SR aM AV where S is the device surface area a is the calibration constant M4 is the analyte molecular weight and V is the analyte volume A R A FE However this would involve modifying the measured d
48. cess commonly used KE functionality Activity Bar items are enabled or disabled based on the currently displayed file If the displayed file does not support a certain action for example you cannot run the Analysis Wizard on a measurement file the item will be disabled New Overlay New Project New Calibration New Van t Hoff Project fi New Subcurve Overlays 1 Proyects 1 Calibrations 1 Vanf Hott Project View Interaction Plot View Data Points View History Analysis Wizard Calibration Wizard Conversion Too Modeling Wizard Signal Processing Vant Hoff Wizard Pe FE r Monte Carlo Wizard Figure 2 12 KE Activity Bar The table below provides an overview of the Activity Bar Autolab SPR KE User manual 11 Table 2 5 Activity Bar Items Section Action Description Create New Overlay Launches the Overlay Builder dialog Files Launches the Project Builder dialog New Calibration Launches the Calibration Builder dialog Opens a measurement file from disk The Measurements number to the right of shows the number of opened measurement files Opens an Overlay file from disk The number to Open Files Overlays the right shows the number of opened overlay files Opens a Project file from disk The number to Projects the right shows the number of opened project files Use this to open Van t Hoff Project files as well as normal Project files Calibrations Opens a Calibration file from disk The n
49. ck the results of the fit for consistency and also to validate the error estimates for the fit Under the Monte Carlo technique the fit is run multiple times under differing conditions The result of each run of the fit is then compared to the original fit Kinetic Evaluation has a Monte Carlo Wizard that can generate two types of Monte Carlo simulations 1 Vary initial parameters for each run of the Monte Carlo the starting points of the fit are varied by a percentage fraction of the fit values The fits are then tested to see if the fit converges to the same final fit values 2 Add noise to the data for each run of the fit a random amount of noise is added to each data point Because the data is modified the fit will typically converge to a different result However the average value of all of the Monte Carlo fits should be consistent with the original fit value 8 2 Running the Monte Carlo Wizard To run the Monte Carlo wizard select an existing analysis within a project Select Monte Carlo Wizard Autolab SPR KE User manual KIN101 5_differential kpf New Overlay New Project New Calibration New Van t Hoff Project New Subcurve Open Files A Measurements Overlays Projects 1 Calibrations Van f Hoff Project Views A View Interaction Plot View SPR Curves View Data Points View Summary Analysis Wizard Calibration Wizard Conversion Tool Modeling Wizard Signal Proce
50. contains functionality to show hide various components of the KE application The screenshot below shows the View menu The View menu contains different items depending on the file type being displayed Figures 2 6 2 7 and 2 8 are screenshots of the three different View menus for each file type File Data Points Channel 2 Differential Event Log Toolbar Activity Bar Options Figure 2 6 Main Menu gt View Measurement file The table below provides an overview of the View menu items Table 2 2 Overview of the View Menu Items Menu Item Description Additional 6 Autolab SPR KE User manual Legend is not Show Hide the graph legend available for Analysis files Measurement files Chart Properties Show the Chart Properties dialog do not have chart properties See Section 4 6 Shows the data of the file in a graph Interaction Plots Measurement files have the option to See Section 4 2 1 show hide plots Show a grid containing the files data See Section 4 2 2 points Event Log Depending on the file type this view See Section 4 2 3 History shows additional data about the file Summary Show Hide the Toolbar See Section 2 3 Show Hide the Activity Bar See Section 2 4 Options Opens a dialog to allow the user to reset the application settings The View menu for an Overlay file provides access to the Chart Properties as shown in the figure below File Tools Hel
51. ct Figure 4 23 Options to Create a new Project If there are Overlay files opened in KE they can be selected in the combo box and imported into the new Project If there are no Overlay files opened then it is possible to import an Overlay file from disk Van t Hoff Projects are a special form of projects that support temperature based analyses These projects are discussed in section 7 Upon clicking the OK button on the Project Builder dialog a new project file is displayed as shown in the Figure 4 24 48 Autolab SPR KE User manual Kinetic Evaluation Jog File View Tools Help D e fel Gil A Pointer zoom Legend Properties EQUI010 1B0 EQUI011 1B0 EQUIOIZIBO EQUI013 1B0 EQUIDI4 IBO Overlay kod Project kp amp Project kpf Create Files A Overlay1 kod New Overlay New Project New Calibration New Van t Hoff Project Measurements 5 Overlays 1 Projects 1 Calibrations Van f Hoff Project H w n E a wo a id View Interaction Plot View Data Points View History Analysis Wizard Modeling Wizard Signal Processing 6000 10000 Time s Figure 4 24 The new project file When a new Project is created the Project Navigation Bar see section 2 5 is displayed on the left hand side of the screen This navigation bar tool is required because Project files can contain multiple sets of data 4 5 2 Performing an Analysis Analysis files are creat
52. ct the overlay to analyze from a drop down combo box as shown in Figure 7 6 Analysis Wizard Step 1 of 7 ole Legend Pointer 4 Zoom Select the Analysis to Launch G New Analysis Create a new analysis for the selected project Figure 7 6 Selecting the overlay for a new analysis in a Van t Hoff Project For the second and subsequent analyses the wizard will either allow the user to create a new analysis from a selected overlay file or to open a previously created analysis as shown in Figure 7 7 118 Autolab SPR KE User manual Legend Pointer j gt Zoom Select the Analysis to Launch New Analysis Create a new analysis for the selected project A Open an Existing Analysis Equilibrium 1 v n Z o a n v oe 1500 Time s Figure 7 7 Selecting to either create a new analysis or open an existing analysis in a Van t Hoff Project The equilibrium analysis process must be repeated for each overlay file in the Van t Hoff Project file Figure 7 8 shows the results for the project after this process is complete There are seven overlay files and seven equilibrium analyses File View Tools Help VantHoffProject_eq kvf Overlay_temp_1 kod Overlay_temp_2 kod Overlay_temp_3 kod Overlay_temp_4 kod Overlay_temp_5 kod New Van t Hoff Project Equilibrium i E quilibrium1l Equilibrium2 Equilibrium3 Equilibrium4 Equilibrium Equilibrium Equilibrium
53. d times Ensure that the Same Region for all Plots option if selected Now select any one of the start times with a single click and then select the Line tool as shown in the figure below Legend Ie Pointer Zoom Line Figure 3 17 Select the Line Tool to Specify the Region With the Line Tool selected mouse over the graph to guide the line to the desired start point and then right click to set the start time The screenshot below illustrates the start time selection Changes made via the Line Tool are reflected in the table and vice versa The start and end times are shown with diamond arrows on the analysis curves Once the start point has been selected the default end point can be used for this example click the Next button Analysis Wizard Step 3 of 7 oeg Set the Region to Analyze Response m Region Options G Same Region for All Plots Unique Regions for Each Plot Show In dR dt vs t Figure 3 18 Using the Line Tool to Select the Start Point e Step 4 of 7 The graph will now show only the region that was selected in the previous step see Figure 3 19 This example will be using the Integrated Rate Equation method Make sure that this option is selected and click Next 26 Autolab SPR KE User manual Analysis Wizard Step 4 of 7 og Legend amp Pointer yp Zoom Select the Analysis Method R Integrated Rate Equation Rate equations that are integrated before the fitting proc
54. e Carlo X Monte Carlo runs based on numerically integrated fits Jj can take longer than those based on integrated models Chose the number of iterations appropriately C Don t show this dialog again Figure 8 3 Numerical Integration warning If the original fit was run against a numerical model that cannot be found by KE the user will see this warning Cannot Find Model A This analysis was created using a numerical integration model KE cannot find the numerical integration model used to create this Fit The Monte Carlo analysis cannot continue Figure 8 4 Cannot find model The Monte Carlo wizard cannot continue The user should refit the data using the Analysis wizard before attempting to use the Monte Carlo wizard 8 3 Run the Monte Carlo The second panel of the Monte Carlo wizard is shown in Figure 8 5 The Wizard shows one histogram for each parameter in the fit In this example there are three floated parameters in the fit Ks E and R Q For each run of the Monte Carlo an entry will be added to the histogram for each parameter The table on the left shows one tab for each plot that is fit In the table are listed the three parameters of the fit along with the original fit value the average of all Monte Carlo runs and the standard deviation of the Monte Carlo runs Autolab SPR KE User manual Once the user clicks the Start button the Monte Carlo will commence
55. e Temperature Variation Threshold the analysis will not be used by default This means that the nclude column will be unchecked Van t Hoff Analysis Wizard Joe Legend Pointer 4 Zoom f 7Select Line Select Analyses for Van t i Hoff Analysis HOTH OTOO g 1e 07 2e 07 3e 07 4e 07 5e 07 6e 07 78 07 8e 07 Concentration M Autolab SPR KE User manual 121 Figure 7 10 Initial panel of Van t Hoff Wizard If the user adjusts the Temperature Variation Threshold the wizard will automatically exclude measurements whose standard deviation is larger than the threshold In Figure 7 11 the threshold has been set to an arbitrary low number and all the analyses have been automatically excluded Van t Hoff Analysis Wizard Joe Legend Pointer 4 Zoom f Select Line Select Analyses for Van t Hoff Analysis 18 07 28 07 38 07 48 07 5e 07 6e 07 7e 07 8e 07 Time 5 Figure 7 11 Adjusting the Temperature Variation Threshold may exclude overlays whose temperature standard deviation shows too much variation Because the Van t Hoff Analysis requires four K4 data points to over constrain the three constants in the fit if the user includes less than four analyses the Next button will be disabled as shown in Figure 7 12 122 Autolab SPR KE User manual Van t Hoff Analysis Wizard Joe Legend Pointer 4 Zoom 11 Select Analyses for Van t Hoff Analysis 180 160 ob O o Include O O oO Som
56. ed Chi Number Iterations Iterations Parameter Value ks 0 0045 E 62 66785 0 Figure 5 51 Integrated Rate Equation Parameters 86 Autolab SPR KE User manual Monophasic Model Chi Squared Chi Number Iterations Iterations Parameter Value b 05 9767 k1 6 3837E 5 k 1 0 0007 Figure 5 52 Numerical Analysis Parameters In certain numerical models such as mass transport the forward and reverse rate equations may need to be kept equal The fit can be forced to fix one parameter equal to the value of another parameter This can be accomplished by setting the value of the second parameter sign and the name of the first parameter to which it should be fixed This is shown in Figure 5 53 405446546 Chi Squared 1 5276E 3 Number Iterations 14 Parameter Value b 04 5062 k F O1SSE F k 1 k1 k2 1 6854E 6 k 2 0 0038 Figure 5 53 Fixing a numerical fitting parameter In this case the fit will be evaluated with K 1 always having the whatever value the fit finds for K1 To start the fitting process after the initial parameters have been tested click the Run button During the fitting process the model plots green lines will be updated on the graph for each successful improvement in fitting to the experimental data The Advanced button is used to configure the parameters of the fitting routine The Advanced Setting dialog shown in Figure 5 54 consists of the following paramete
57. ed by the Analysis Wizard which is described in section 5 3 Analysis files are a part of a Project file and cannot be saved separately The reasoning for this restriction is to guarantee that the analysis is always sorted with the Overlay file also stored in the Project file was used for the analysis The result running the Analysis Wizard is that a new analysis will be added to the project The project now handles three different types of analysis Association Equilibrium and Dissociation analysis 4 5 3 Navigation of a Project File Project files contain one Overlay file and analysis files It 1s possible to view the different components of the Project file by clicking on them in the Project Navigation Bar Figure 4 25 shows the Project Navigation Bar in which an analysis named Associaton is selected Autolab SPR KE User manual 49 Project kpt Overlay kod Association a Figure 4 25 Changing the Visible Plot 4 5 4 Types of Project Files In KE 5 1 there now exists three types of project file The preceding section has discussed a default project file One can also create Calibration projects and Van t Hoff projects The differences in these project types are detailed in Table Table 2 1 Overview of the File Menu Items Project Types Purpose Allowed Number of Additional Analyses Overlay Files Project Data analysis Association One Overlay File Section 5 3 Dissociation only Equilibrium only
58. eees 124 8 2 1 Monte Carlo Analysis of Numerically Integrated Fits 126 SF Run the Mone Carl Oera rena gene EN 126 8 5 1 Vary the Start Parameters 24 DEERE 129 8 3 2 Add Noise to the Data 0 W W ssssssseeseersessessseseeneenessssseneennsssssenneenesseeee 129 584A Mone Calo S NAY esere EE Ei 130 Autolab SPR KE User manual p lt Autolab SPR KE User manual Kinetic Evaluation 5 2 1 Introduction Kinetic Evaluation KE is the data analysis application that is bundled with the Autolab SPR Software This version of KE has been redesigned incorporating the functionality of the previous versions of KE while introducing new analysis techniques and improving the overall workflow of the system The remaining sections of this manual are divided into the following topics e Section 2 Working with KE This section describes the components that make up the user interface of the Kinetic Evaluation application e Section 3 Getting Started with the KE Workflow This section provides an example of a typical workflow with the software This example starts with opening data files and proceeds to fitting interaction plots using the Analysis Wizard e Section 4 Working with Data An overview of all the file types that the KE application can open create and save e Section 5 Analyzing the Data Description of the available analysis tools within the KE application The Modeling Wizard used to create
59. el 1 KIN102 Channel 1 KIN1 4 gt Include this plot Chi Squared w oOo N n Response m 1110 171 1 800 Time 5 Figure 3 22 Initial Plot of the Fit Parameters Once the initial fit parameters have been tested the fitting of the data can be performed by clicking the Run button During the fitting procedure the plot that is currently being fit will be shown in the tab on the left of the screen The green model lines for the plots will be 28 Autolab SPR KE User manual redrawn upon each iteration of the fitting routine that improves the Chi of the fit Figure 3 23 depicts the fitting of Plot 2 A more detailed overview of the fitting procedure can be found in section 5 3 Once the fit procedure has ended click Next to proceed to the summary Analysis Wizard Step 6 of 7 oE Legend Pointer 4 Zoom Configure and Fit the Model Plot 1 Plot 2 Plot 3 Plot 4 Plot 5 Include this plot Parameter Value 0 0023 252 6452 36 8176 tN h o Response m ho 3 h o Advanced 600 800 Time s Figure 3 23 Running the Fitting Procedure h Step 7 of 7 The final step of the Analysis Wizard is the summary of the performed fit The summary as shown in the figure below shows the general configurations that were used and the final parameter values for each plot This data can be copied to the clipboard by clicking the Copy to
60. enone seen ttececciomns saessceemencecsecenpencess 104 6 2 The Calibration W1Zard cccccssccccccecccccceeeeeeaeeeeeeeesseeeeeeeeeeeeeeeeeeeeeaaqqaas 105 6 3 Specifyins the Concentrations 222 ane bre ea ae eg eee a eia ionas 106 6 4 Specifying the Start End Times of the Analysis cccccccccssseseeeeeeeeeeeeees 106 6 5 Extrapolation to Equilibrium cc cseeceseccccceccaeeeeeeeecceeeeeaeeeeeeseeeeeeeeeaas 107 OT Analy iS e eatin de re ere a 108 BT GUT FASE SEE E E T E E E E EE 109 UMM A castes E E 109 Or sie Calibration TICS sisses an A ESETE AE 112 7 Van t Hoff Analysis Wizard cccccccsssssesesseseeseeeseeeeeseeeeeeeeeeeeeseeeeeeeeeseseeeeeeees 113 e a S E E 113 7 2 Creating a Van t Hoff Project ccccccscsssssseseseseeseseseeeeeeeeeeeeeeeeseeeeeeeeeseeees 113 fe eh C00 018 MUA Lg ANC Blk hs Geen ee aero ee eer ee errr ener entre ne een ERE errr ere een ere 114 7 4 Saving and Loading Van t Hoff Projects ccccccccccccceeeeeeeeeeeeeeeeeeeeeeees 115 7 5 Van t Hoff Equilibrium Analysis cccccccccccccccccecceeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 116 1 6 Van t Hoff Analysis Wizard ea cepted aera ce eased ease cieeesacueenennsees 120 Autolab SPR KE User manual 5 Mofite Carlo Wizard sonarer need erne eba e 124 etl TR 6 HEH SEERE E TEE 124 8 2 Running the Monte Carlo W1Zard cccccccssseessecccccceeeeeeseeecceeeessaaaeeseeee
61. ental data If you do not wish to see the information dialog the next time the wizard is run click the Don t show this dialog again checkbox Autolab SPR KE User manual 85 Instruction x In this step the actual takes place For best results the initial fitting parameters can be plotted by pressing the Test button Once the initial values are close to the data plots press the Run button to start the fitting Don t show this dialog again Figure 5 50 Additional Instructions for Fitting Data The configuration of the fitting parameters for the Integrate Rate Equation method see Figure 5 51 and the Numerical method see Figure 5 52 are quite similar The difference between the two methods is that the Integrate Rate Equation method uses a Separate set of fitting parameters for each interaction plot while the Numerical Modelling method uses one set of parameters for all plots Numerical Modelling uses the Global Analysis technique in which all of the plots are fit at the same time to the same set of parameters values The benefit of using global analysis is that it ensures that the selected model does in fact fit to the experimental data because the binding constants are the same in all interaction plots only the concentrations differ The initial values of the parameters are shown in Figure 5 52 Configure and Fit the Model Plot 5 Plot 4 Plot 3 Plot 2 Plot 1 Include this plot Chi Squar
62. ess O Numerical Modeling Complex rate equations that are resolved by numerical calculations during the fitting process Response m N 5 600 800 1000 1200 1400 Time s Figure 3 19 The Graph now shows only the selected region f Step 5 of 7 This example will be using the Monophasic Integrated Rate Equation Ensure that this method is selected as shown in the figure below and click Next Select the Model to Use Monophasic Monophasic ps Biphasic Simple 1 1 interaction model Figure 3 20 Selecting the Monophasic Model g Step 6 of 7 In this step of the Analysis Wizard the fitting of the model to the experimental data will take place It is strongly suggested that the initial fitting parameters be tested before starting the fit procedure To test the parameters press the Test button as shown in the figure below Autolab SPR KE User manual 27 Configure and Fit the Model KIN101 Channel 1 KIN102 Channel 1 KINI 4 gt Include this plot Chi Squared Number Iterations saa Figure 3 21 Testing the Initial Fit Parameters The initial fit parameters are acceptable if the model plots green lines are of the same order of magnitude as the experimental data Figure 3 22 shows the initial fit plot for this example which 1s acceptable Analysis Wizard Step 6 of 7 Jo Legend R Pointer 4 Zoom Configure and Fit the Model KIN101 Chann
63. first panel of the Analysis Wizard as shown in Figure 5 69 Autolab SPR KE User manual 99 Select the Region to Analyze Association Phaze in the interaction plot which the analyte combines with the immobilized ligand to form a complex Equilibrium Phase in the interaction plot where the observed rate of formation for the complex is zera Dissociation Phaze in the interaction plot where the analyte is removed from the immobilized ligand Determine Concentrations Determine the concentrations in the overlay by applying a calibration file Figure 5 69 Select Determine Concentrations The analysis wizard will proceed in four steps Each step is similar to a step in the Equilibrium analysis but the steps occur in a different order Because setting the concentrations from a calibration curve uses an equilibrium analysis the user should not use this method to set the concentrations if they then want to perform an equilibrium analysis see section 5 3 2 Doing so will simply return the equilibrium analysis that was initially used to create the calibration curve 5 3 14 Setting the Region to Analyze for Determining the Concentrations The concentrations are determined by fitting each of the measurements via an equilibrium analysis Therefore the next step is to set the region to fit as shown in Figure 5 70 Knalysis Ward Step Dor 4 Sex Set the Region to Analyze Legend Pointer gt Zoom Li
64. gnal Processing 0 1 2 3 4 5 6 7 8 9 Time s Figure 2 1 Screenshot of the Kinetic Evaluation application The following descriptions correspond to the numbers that appear in Figure 2 1 1 Opened Files Files that have been opened are added as tabs The data within these files are graphed within the tabs For more information about the file types that KE supports see section 4 2 Main Menu All the functionality of the KE application is accessible via the main menu see section 2 2 for more information 3 Toolbar The buttons in the toolbar represent KE functionality see section 2 3 for more information 4 Activity Bar Provides quick access to commonly used functionality The functions in the Activity Bar are grouped into common themes see section 2 4 for more information Autolab SPR KE User manual SD 5 Project Navigation Bar Provides a method to view the data that is stored in a Project file see Section 2 5 for more information This Navigation Bar is only displayed when a Project file see section 4 5 is in focus 2 2 Main Menu The main menu provides functionality to access all the features of KE Figure 2 2 below is a screenshot of the Main Menu File View Tools Help Figure 2 2 Main Menu of KE The following subsections describe the Main Menu items 2 3 File Menu The File menu contains functionality that is required to work with files in the KE applicatio
65. h tab ee neem shows the Contents tab shows the Index tab Evaluation number Autolab SPR KE User manual 9 2 4 Toolbar The Toolbar contains buttons that launch KE functionality The screenshot below illustrates the Toolbar a EA a Pointer Zoom i 1Select Legend ff Properties Figure 2 11 KE Toolbar The following gives an overview of the items in the Toolbar L oo amp k Pointer gt zoom rt Select Legend Froperties os Creates a new Overlay or Project file depending on which type the user chooses Opens a Measurement Overlay or Project file depending on which type the user chooses see section 4 1 2 Saves the file in the visible tab Saves all the open files The default mode of the graph When moving the mouse pointed over the graph the coordinates x y will be displayed next to the mouse icon Sets the graph to Zoom mode Left click and dragging the mouse pointer will zoom in on the selected region Sets the graph to Select mode Left click and dragging the mouse pointer will select an area of the graph Shows Hides the graph legend Displays the Chart Properties dialog see Section 4 6 Creates a sub curve from the selected area of a measurement file see Section 4 3 2 F Manage Overlays Launches the Manage Overlays dialog see Section 4 4 1 10 Autolab SPR KE User manual 2 5 Activity Bar The Activity Bar as shown in Figure 2 12 is used to ac
66. hat assists the user in analyzing data The figure below gives a picture of Step 1 which is displayed when the wizard is opened Selected the Association option and click Next 24 Autolab SPR KE User manual Analysis Wizard oE Select the Region to Analyze Legend Pointer 4 Zoom Association Phase in the interaction plot which the analyte combines with the immobilized ligand to form a complex Equilibrium Phase in the interaction plot where the observed rate of formation for the complex is zero Dissociation Phase in the interaction plot where the analyte is removed from the immobilized ligand Response m an oOo Determine Concentrations Determine the concentrations in the overlay by applying a calibration file o 1500 Time s Figure 3 15 Analysis Wizard c Step 2 of 7 In this step the concentration for each of the measurements is entered The screenshot below shows the values that are to be entered Fill out the concentrations and click Next Refer to Section 6 for a discussion of using Calibration Files to set the concentrations Enter the Plot Concentrations KIN101 Channel 1 KIN102 Channel 1 KIN103 Channel 1 KIN104 Channel 1 KIN105 Channel 1 Figure 3 16 The Concentrations for the Measurements Autolab SPR KE User manual 25 d Step 3 of 7 Selecting the Analysis Region For this analysis all plots are going to use the same start and en
67. icking OK The Van t Hoff project will then be displayed in the main KE window The Project Navigator shows the new Van t Hoff project VantHoffProject1 kvf with the five imported overlays Each overlay corresponds to measurements taken at a single temperature The Van t Hoff project is the only project type where multiple overlay files can be imported into one project Autolab SPR KE User manual 115 Kinetic Evaluation Fie View Tools D LA g Zoom E Legend pf Properties Overlay_temp_1 kod Dverlay_temp_2 kod Overlay_temp_3 kod Overlay_temp_4 kod Overlay_temp_5 kod VantHoffProject_e gt 5 VantHoffProject_eq kvf Create Files Overlay_temp_1 kod Overlay_temp_2 kod erla Overlay_temp_3 kod New Project Overlay_temp_4 kod New Calibration Overlay_temp_5 kod New Van t Hoff Project Open Files A Measurements Overlays 5 Projects View Interaction Plot View Data Points View History Analysis Analysis Wizard Modeling Wizard Signal Processing Van t Hoff Wizard 1500 Time s Figure 7 3 The Van t Hoff Project in the main KE window At this point one could execute Signal Processing on each of the overlay files 1f necessary Signal Processing is discussed in Section 5 2 7 4 Saving and Loading Van t Hoff Projects Van t Hoff Projects are saved with a kvf file extension These project files can be loaded from the Open Files Activity bar by
68. ing technique and how the technique will be applied There are two options for how the technique will be applied it can process the entire plot or only a portion of the plot that is selected via the Select mode click the Select button and with the right mouse button down drag the mouse to select a region 3 Apply Undo Once the technique and the plots that will be processed have been configured press the Apply button to run the action If the action did not Autolab SPR KE User manual 61 perform the desired effect then this action can be undone by pressing the Undo button There is no limit to the amount of times an action can be applied 4 Overlay Data The overlay data is updated upon each press to the Apply button All signal processing changes can be viewed in this graph 5 Graph Toolbar The figure below depicts the tools that are available for the Signal Processing dialog Legend k Pointer zoom 1 Select Figure 5 13 Signal Processing Toolbar The Toolbar contains the following items Table 5 1 Toolbar Items Toolbar Item Description 00000 Legend Show Hide the legend for the Overlay Pointer This is the default mode of the graph a tescecediegen will zoom in on the selected region ER region for the signal processing action to process The following subsections will give an overview of each of the Signal Processing techniques 5 2 1 Blank Subtraction
69. ired time Click single click to set the time Figure 5 43 highlights the items that are used in this method Region Options G Same Region for All Plots Unique Regions for Each Plot Show In dR dt vs t Figure 5 43 Using the Line Tool to Select the Start Time Autolab SPR KE User manual 81 It 1s possible to obtain additional information about the association starting point of the curve Selecting Unique Regions for Each Plot and Show In dR dt vs t will overlay the logarithm of the derivative versus time and can be useful to see mass transport effects This option is only available when unique regions are used for each plot The user has to set each starting and ending point for each plot The overlay of the derivative curve is shown in Figure 5 44 Analysis Wizard Step 3 of 7 MIS Legend Pointer 4 Zoom Line Set the Region to Analyze nm c en gt E D xr ER 2000 2500 Figure 5 44 Showing the derivative of the curve to aid in setting the Start Time 5 3 4 Selecting the Analysis Method The next step in the Analysis Wizard is to select the method that will be used for the fitting of the data Figure 5 45 depicts a screenshot of this step This Analysis Wizard step is not required for Equilibrium Analysis For Equilibrium Analysis skip to section 5 3 8 The current Kinetic Evaluation application provides two analysis methods Integrated Rate Equa
70. is to use a random number generator to add a random amount of noise to the actual data Here the data points are changed The noise is selected from a Gaussian distribution with a standard deviation equal to the noise factor input by the user Figure 8 8 shows the result of a noise Monte Carlo where 10 millidegrees of noise was added to the data The fits show that the Monte Carlo average is very similar to the original fit values The standard deviation of the Monte Carlo values is larger than the estimated uncertainties on the original fit due to the addition of the 10 millidegrees of noise in the data 130 Autolab SPR KE User manual Run Monte Carlo Legend Pointer Run 100 of 100 Parameter Original Value MC Average MCS 0 0018 0 0018 2 286 214 0621 214 0615 0 807 9 0484 9 0491 0 690 Figure 8 8 Monte Carlo results for noise fit 8 4 Monte Carlo Summary The final panel of the Monte Carlo wizard shown in Figure 8 9 summarizes the Monte Carlo run Monte Carlo Summary Legend R Pointer Summary Number of Iterations 100 Number of Plots 5 Variation 100 _KIN101 Differential KIN102 Differential KIN 4 gt Parameter Original Value MC Average MC S Ks 0 0018 0 0017 0 000 E 214 0621 208 2829 42 02 R 0 9 0484 10 4799 10 13 FE AN AN RE HE i AE a OSE 005 0 001 ne escuela Figure 8 9 Monte Carlo Summary Panel Autolab SPR
71. ium will merely return the results inthe Calibration analysis C Don t show this dialog again Figure 5 40 Equilibrium concentration warning Because the concentrations were determined from a Calibration curve and the calibration curve uses an equilibrium analysis see section 6 1 running the equilibrium analysis will simply return the same analysis from the calibration analysis 5 3 3 Specifying the Start End Times of the Analysis The next step in the Analysis Wizard is to specify the start and end times for the type of analysis region that was selected In general the following guidelines should be followed when selecting the start end times Autolab SPR KE User manual 79 e Association The start time is the start of the positive sloped curve while the end time is where the slope levels off towards zero e Equilibrium Use the same starting point as Association and the end time will be the time right before the slope changes to a negative value this is the start of Dissociation e Dissociation The start value is the start of the region with a negative slope while the end region is where the slope levels off to zero To select the start and end times the following instruction dialog will be presented to the user Instruction There are two ways to setthe analysis region 1 Select the startend value that vou wish to change and toe in the new value 2 Select the startend value that
72. kinetic models the Signal Processing tool used to improve data quality and the various Analysis Wizards used to determine kinetic binding constants e Section 6 Calibration A description of calibration curves that can be used in analyses e Section 7 Van t Hoff Analysis A description of analyses versus temperature that measures thermodynamic properties 2 Autolab SPR KE User manual 2 Working with KE In this section introduces the user interface of the KE application The navigation via various menus and toolbars will be described in the following subsections 2 1 User Interface Overview Figure 2 1 is a typical screenshot of the KE application In this section each of the numbered regions in the figure will be explained Kinetic Evaluation BAX File view Tools Help Deag amp Pointer 4 gt Zoom Legend p Properties 3 KIN101 5 kpf Calibration kcf KIN101 1B0 KIN102JBO KINIO3 1IBO KIN104 1B0 KINIO5 1IBO Overlay1 kod Project1 kpf 4 gt amp A B A4B kpf l Ao 4___ 3Plots kod 3 2 PEE Association Association New Overlay 3 Association2 Dissociation Dissociation 2 6 5 New Project New Calibration New Van t Hoff Project 2 8 Open Files A Measurements 5 Overlays 1 Projects 3 Calibrations 1 Van f Hoff Project Response m View Interaction Plot View Data Points View History Analysis Wizard Modeling Wizard Si
73. model Select the Model to Use Monophasic Monophasic Biphasic Simple 1 1 interaction model Figure 5 46 Selecting the Integrated Rate Equation If you have selected Dissociation analysis a third model option will appear Rebinding Monophasic as shown in Figure 5 47 This is a monophasic model that incorporates terms related to rebinding See for example Schuck and Minton Analysis of Mass Transport Limited Binding Kinetics in Evanescent Wave Biosensors Analytical Biochemistry 240 262 272 1996 Autolab SPR KE User manual 83 For the fit to the monophasic rebinding model to converge it is necessary for the user to supply some input parameters Bmax and Ka Ki see the Schuck and Minton paper for definitions Bmax can be determined from an equilibrium analysis and if equilibrium analyses have been performed it can be imported from an opened equilibrium analysis by using the combo selection box The box is not shown if equilibrium analyses have not been performed Ka Ky is typically a small number lt lt l Select the Model to Use For the fits to converge you must supply values for Bimax and k aj k tr If you have run equilibrium analyses you can select B max from one of those analyses Otherwise an estimation of B max is provided Rebinding Parameters Bim om Bimax 9 4266E 8 kalki Figure 5 47 Selecting the Integrated Rate Equation Selecting a model for the Numerical
74. mum value from the data is greater than the fit maximum value The fit threshold can be changed in the text box if necessary 92 Autolab SPR KE User manual Set Plots Not At Equilibrium KIN101 Channel 1 KIN102 Channel 1 KIN103 Channel 1 KIN 104 Channel 1 KIN105 Channel 1 Threshold to consider curve at equilibrium 0 01 Figure 5 61 Table of equilibrium plots before fitting for equilibrium Once the Fit button is clicked each curve will be fit for equilibrium and the results presented as shown in Figure 5 62 In the figure the maximum from the fit is shown in the column next to data column If the fit indicates that the plot is not at equilibrium the Not at Equilibrium checkbox will automatically be checked This is shown in the area marked 1 in Figure 5 62 Furthermore the extrapolated fits will be shown in the area marked 2 in Figure 5 62 This is shown to aid the user in deciding the goodness of fit If the user believes the plot is not at equilibrium the user can check the Not at Equilibrium checkbox to use the value from the fit instead of the value from the data Autolab SPR KE User manual 93 a Analysis Wizard Step 4 of 7 oE Legend Pointer 5 Zoom Set Plots Not At Equilibrium gt KIN101 Channel 1 KIN102 Channel 1 KIN103 Channel 1 323 16 KIN104 Channel 1 337 08 KIN105 Channel 1 357 18 Nm oO oO Response m 0 01 1500 2000 2500 3000 3500 Time s Figure 5 6
75. n The screenshot below depicts the File menu View Tools Help Pe Open Clase Close All Save Save Projectz kpf As Save All Prink Preview Print Exit Figure 2 3 Main Menu gt File The table below on this page provides an overview of the File menu items Table 2 1 Overview of the File Menu Items Menu Item Description Additional New gt Overlay Displays the Overlay Builder dialog Figure 2 4 Section 4 4 Section 4 5 1 dialog Section 4 5 2 4 Autolab SPR KE User manual Section 4 3 Section 4 4 lees Van t Hoff Section 4 5 Section 4 E OS 2 Closes the file in the visible tab Close All Closes all open files Saves the file in the visible tab Save As Saves a copy of the opened file with a new name Exit Exitsthe KE application The File 2New menu item contains sub menu items as shown in Figure 2 4 To access these items click the New menu item and the sub items will appear Project X Calibration Save Project2 kpf As c amp Print Preview ER Print Exit Figure 2 4 File gt New Menu The File 2Open menu item also contains sub menu items Figure 2 5 Autolab SPR KE User manual 5 Measurement Overlay Project nduding Van t Hoff Calibration Save Project2 kpf As Bi cL Print Preview ER Print Exit Figure 2 5 File gt Open menu 2 3 1 View Menu The View menu item
76. n Existing Model from disk Select the Create New Kinetic Model and click Next 5 1 2 Configuring the Modeling Information 54 Autolab SPR KE User manual Step 2 of the Modeling Wizard is shown in Figure 5 2 In this step the information about the model is entered Every model is given a name and an optional description Modeling Wizard Step 2 of 5 Wl Start Model Name i Click to add a model name HH Model Information O Define Species Model Description O Equation Editor O Summary Figure 5 2 Model Information Step To give the model a name click the text Click to add a model name To add a description to the model click in the Model Description text box and enter the text Figure 5 3 shows an example of a model name and description entered Add your model name and description and click Next Modeling Wizard Step 2 of 5 Wl Start Model Name E Model Information Monophasic Model LJ Define Species Model Description O Equation Editor Standard 1 1 interaction model O Summary Figure 5 3 Information for the Model Autolab SPR KE User manual 55 5 1 3 Defining the Species In Step 3 of the Modeling Wizard the species that are used in the rate equations will be defined Enter in each unique species for the rate equation as shown in Figure 5 4 Once you have entered in your species click the Next button to proceed fw Start Define Species be Model Information Specify the names
77. n Figure 4 3 Select the Type of File to Open Measurement File containing SPR data spr ksc ibo tet Overlay File containing multiple plots kod Project File containing experiment data or Vant Hoff analysis kpt kvf Calibration File containing calibration data ket Figure 4 3 Opening an Overlay file from the Toolbar 4 1 3 Opening a File from the Activity Bar The third method to open data files in KE is to click on the file type in the Activity Bar Figure 4 4 depicts the action of opening a Project file Autolab SPR KE User manual 33 New Overlay New Project New Calibration New Van t Hoff Project New Subcurve Overlays er Cali Vanf Hoff Project Figure 4 4 Opening a Project file from the Activity Bar 4 2 Viewing Files The KE application allows the user to view all files Measurement Overlay Project and Calibration using three different views Changing the current view for a file can be performed via the Main Menu see Figure 4 5 or by clicking on the desired view from the Activity Bar as shown in Figure 4 6 Kinetic Evaluation Tools Help tz Legend Legend Properties Chart Properties Interaction Plot Data Points History Toolbar Activity Bar Options Figure 4 5 Changing the View Mode via the Main Menu 34 Autolab SPR KE User manual View Interaction Fl View SPR
78. n m o X Fie view Tools Help a Zoom Legend g amp Properties KIN101 5 kpf Calibration1 kcf Calibration kef Overlay1 kod New Calibration New Van t Hoff Project Open Files A Measurements Overlays Projects 1 Calibrations 1 Van f Hoff Project M am Views A _ mn Oo a no a n a oe View Interaction Plot View Data Points View History Calibration Wizard Signal Processing 1500 Time s Figure 6 3 A newly created calibration project To launch the Calibration Wizard select Calibration Wizard from the Activity bar as shown in Figure 6 4 106 Autolab SPR KE User manual Calibration neah Signal Processing Figure 6 4 Launching the Calibration Wizard from the Activity bar The Calibration Wizard is similar to the Analysis Wizard see section 5 3 but it only runs a simplified equilibrium analysis 6 3 Specifying the Concentrations As with the Equilibrium analysis the first step 1s to set the concentrations as shown in Figure 6 5 Calibration Wizard AA Enter the Plot Concentrations Legend Pointer j gt Zoom Concentration Unit KIN102 Channel 1 25 KIN101 Channel 1 100 KIN103 Channel 1 200 KIN104 Channel 1 400 M O O KIN105 Channel 1 500 Response m oOo 1500 Time s Figure 6 5 Plot Concentrations for the Calibration Wizard 6 4 Specif
79. nce the concentration calculation is completed the calculated concentrations will be shown in the table circled in Figure 5 75 In addition the calibration curve will be shown with blue data points for the original calibration curve data and pink points 102 Autolab SPR KE User manual for the calculated concentrations This plot is shown in more detail in Figure 5 76 after the Legend button in the Toolbar was pressed ar Wizard Step 4 of 4 Joes Enter the Plot Concentrations Legend Pointer g gt Zoom Use Calibration to Set Concentrations Open Calibration File C Documents and Settings Jim Use existing calibration Concenh Unit Maimun i gt Shera 288507 M 228 76 Ditorenta 34 8914 254 44 Dimona 541080 gM 260 8 Ditorenta 66 4391 x 230 96 1e 07 2e 07 3e 07 5e 07 6e 07 7e 07 8e 0 4e 07 Concentration M Figure 5 75 Calibration after calculating the concentrations Legend Pointer 4 Zoom Csiculsted Concentrations Concentration Calibration Data 350 Regression 300 250 0 2e 07 4e 07 6e 07 8e 07 Concentration M Figure 5 76 Concentration curve showing the calculated concentrations after the Legend button was pressed It is possible that a given calibration curve cannot be applied to the current data This situation can occur when the Reguilibrium Of the extrapolated data from Step 3 is greater than the Bmax of the calibration curve
80. ne Start KIN102 Channel 130 47 1 KIN101 Channel 130 47 1 KIN103 Channel 130 47 1 KIN104 Channel 130 47 1 KIN105 Channel 130 47 1 Response m Region Options Same Region for All Plots Unique Regions for Each Plot Show In dR dt vs t 0 500 1000 1500 2000 2500 Time s Figure 5 70 Set the Start End points for determining the concentrations 5 3 15 Determine plots not at equilibrium 100 Autolab SPR KE User manual The next step is to determine which plots are not at equilibrium By clicking the Fit button as shown in Figure 5 71 the plots will be fit to a set of equilibrium curves However it is not required to try to determine if the curves are at equilibrium if the Max from Data valid the user can elect to continue to the next step by clicking the Next button Pros Wizard Step 3 of 4 JOR Set Plots Not At Equilibrium Legend Painter p Zoom fom hom Notat Data Fiting E9 gt KIN101 Differential 225 85 oO KIN102 Differential 254 49 oO KIN103 Differential 280 8 oO KIN104 Differential 290 96 oO KIN105 Differential 302 13 oO Threshold to consider curve at equilibrium Response m 1000 1500 Time s Figure 5 71 Apply Equilibrium fit to data If the user decides to fit the plots the plot panel will show the data along with a regression curve as sh
81. of 100 KINTOT Differential KIN102 Differential KIN 41 gt Parameter Original Value MC Average MCS Ks 0 0018 0 0018 0 000 E 215 8559 191 9548 239 0 R 0 6 7520 7 2490 4 969 1500 1000 E 50 29 104 4 Figure 8 7 The Monte Carlo run completed Autolab SPR KE User manual 129 8 3 1 Vary the Start Parameters The first option for the Monte Carlo is to vary the starting point of the fit In this option the parameters that are used as starting points for the fit are varied by the specified percentage although they are not varied negative The fits are then run to determine if the final parameters that are determined from the fit are the same as from the original fit This technique allows the user to determine if the original fit found a local minimum of the fit or if the original fit converged to an absolute minimum If all of the varied parameter runs end up converging to the same set of final parameters the user has confidence that the reported parameters are the best fit for the given model It is important to realize that the presence of only one set of final parameters does not mean that the chosen model is a good fit to the data It only means that there is one set of parameters that work for that model This technique is not a true Monte Carlo because the actual data is not modified 8 3 2 Add Noise to the Data The second option for the Monte Carlo
82. ossible to copy this data to another application by clicking the Copy to Clipboard button Summary View Analysis Files When an analysis is performed using the Analysis Wizard see section 5 3 an analysis is added to the project file By changing the view of the analysis to Summary it is possible to view the results of the fitting The fitting results can also be copied to another application by clicking the Copy to Clipboard button Figure 4 13 shows the summary view for an Association analysis 40 Autolab SPR KE User manual Projectl kpf Dverlay1 kod Association Association jeet ay NewCatirion frem arose __ Motoa mogens _ w jessone w eezanea swe bo JIO CE III a tesionePaswo smo I i SS ee oe femman Ut Sit Sn Saad rate Ke Kee KMO Chawelt anqom fem 41 1630 6627 28 oome ezus 18 OO name FE LEM Analysis Wizard Calibration Wizard Conversion Tool Modeling Wizard Signal Processing Van t Hoff Wizard Copy To Clipboard Monte Carlo Wizard Figure 4 13 Summary View of an Analysis Autolab SPR KE User manual 4 4 3 Measurement Files Measurement files are generated using the Autolab SPR Data Acquisition application KE also provides support for measurement files of different formats KE supports the following file types of measurement files Table 4 1 Supported Measuremen
83. ot input the actual winda 8 size but a smooth factor m The wird size is 2 rit Figure 5 27 Smooth Options The screenshot Figure 5 28 is before and Figure 5 29 is after the Smooth action Autolab SPR KE User manual 69 Legend k Painter P Zoom 15elect Response m 50 Time s Figure 5 28 Overlay before Smoothing Legend k Painter P Zoom Select hake Ty eee Pals Flg i W i i ER ge vi T A Poti a i ry ii i A VEN p fi ANEA TAA i o a wn c a Z wn a of 50 Time Figure 5 29 Result of the Smooth Action 5 2 7 Synchronize The Synchronize action removes the selected region between plots so a common starting time can be achieved Typical data in which this action should be applied is shown in Figure 5 30 Notice that the starting times of the association regions do not start at the same time point 70 Autolab SPR KE User manual Legend k Pointer Zoom E 1 Select bl oa o Response m 2 s00 1000 Time Figure 5 30 Association Regions starting at Different Times To synchronize the starting times Zoom in on the starting points of the plots and use the Select tool in the Signal Processing Toolbar to select the region to synchronize Figure 5 31 depicts this selection Legend k Pointer j zoom K 1 5elect 100 F 90 80 Response m 100 Time Figure 5 31 Selecting the Region to Synchronize Ensure
84. own in Figure 5 72 eros Wizard Step 3 of 4 Joe Set Plots Not At Equilibrium Legend IK Pointer Zoom Max Max from from Data Fitting gt KIN101 Differential 225 65 228 71 KIN102 Differential 254 49 256 51 KIN103 Differential 280 8 281 97 KIN104 Differential 290 96 291 67 KIN105 Differential 302 13 302 35 Threshold to consider curve at equilibrium 0 01 Fit Response m 1500 2000 2500 3000 Time s Figure 5 72 After Equilibrium fit Similar as was shown in the Equilibrium Analysis Figure 5 62 the Determine Calibrations analysis will show plots not at equilibrium The Not at Equilibrium checkbox will automatically be checked This is shown in the area marked 1 in Figure 5 72 Furthermore the extrapolated fits will be shown in the area marked 2 in Figure 5 72 This is shown to aid the user in deciding the goodness of fit If the user believes the plot is not at equilibrium the user can check the Not at Equilibrium checkbox to use the value from the fit instead of the value from the data In Figure 5 72 only KIN101 Differential the red plot is considered to be Not at Equilibrium Autolab SPR KE User manual 101 5 3 16 Apply the Calibration file The final step of determining the concentrations is to apply the calibration curve to the equilibrium data This process is shown starting in Figure 5 73 Analysis Wizard Step 4 of 4 JOR Enter the Plot Concentr
85. p Legend Chart Properties Interaction Plot Data Points History Toolbar Activity Bar Options Figure 2 7 Main Menu gt View Overlay file lt Q Autolab SPR KE User manual The View menu for an Analysis file File Tools Help Chart Properties Interaction Plot Data Points Summary Toolbar Activity Bar Options Figure 2 8 Main Menu gt View Analysis 2 3 2 Tools Menu The Tools menu provides access to functionality to analyze data The screenshot below portrays the Tools menu File View Tools Help Analysis Wizard Modeling Wizard Signal Processing Figure 2 9 Main Menu gt Tools The table below provides an overview of the Tools menu items Table 2 3 Overview of the Tools Menu Items Menu Item Description Additional Analysis Wizard Opens the Analysis Wizard dialog See Section 5 3 Calibration Wizard Opens the Calibration Wizard dialog 2 3 3 Help Menu 8 Autolab SPR KE User manual The Help menu provides access to the KE s online help system The screenshot below shows the Help menu File View Tools Search Contents Index About Kinetic Evaluation Figure 2 10 Main Menu gt Help The table below provides an overview of the Help menu items Table 2 4 Overview of the Help Menu Items Menu Item Description Additional pean Om NowstheScachtabe shows the Searc
86. pplication are shown in the screenshot below Notice that there is now the number 5S beside the Measurements item to indicate the number of files opened Kinetic Evaluation m x File View Tools Help yp Zoom 1 iSelect Legend KIN101 IBO KIN102 1B0 KIN103 1B0 KIN104 1B0 KIN105 1B0 Create Files New Overlay New Project New Calibration New Van t Hoff Project Measurements 5 Overlays Projects Calibrations Van f Hoff Project N a w no Z a n a e N gt Temperature C Views View Interaction Plot View SPR Curves View Data Points View Event Log 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 Time s Figure 3 3 Data Loaded in the KE Application 3 2 Creating an Overlay In this step the five measurement files that were opened will be used to create a new overlay file a Click the New Overlay item in the Activity Bar as shown in the screenshot below Autolab SPR KE User manual 17 New Overlay New Project New Calibration New Vant Hott Project New Subcurve Measurements 5 Overlays Calibrations Vanf Hoff Project Figure 3 4 Selecting the New Overlay item b Example of a new overlay using the Channel 1 data from each of the measurement files Select the Channel 1 checkboxes for each measurement as shown in the screenshot of the overlay builder below Once all the Channel 1 s have been selected click
87. re 2 14 the name of the overlay file is A B AB__Splots kod Autolab SPR KE User manual 13 The next groups are the analyses that have been performed on the Overlay data The analyses are grouped into Association Equilibrium and Dissociation analysis The project in Figure 2 14 contains two association analyses one equilibrium and no dissociation analysis 2 6 1 Editing Navigation Bar Items Editing the items in the Project Navigation Bar can be performed by right clicking on the item to display a pop up menu as shown in Figure 2 15 below To rename an item right click on the item and select Rename The item s name will be highlighted and the new name can be entered Project kpt 44 R 4R plots kod Rename K Ki Remove pation Associaton E quilbrium E quilibrivinn Figure 2 15 Renaming a Project Component 2 6 2 Removing Navigation Bar Items It is possible to remove an item from a Project via the navigation bar To remove an analysis it is not possible to remove the overlay right click on the analysis and select Remove from the pop up menu see Figure 2 16 A confirmation will be required to complete the removal Project kpt 4 b 46 plots kod Association Association Association Equilbriurn E guilibrium Rename Remove Figure 2 16 Removing an Analysis from a Project 14 Autolab SPR KE User manual It is not possible to remove the Overlay file from a Project
88. rs Autolab SPR KE User manual 87 e Step Size Step size between iteration e Number Steps Number of steps with defined step size e Max Iterations Maximum number of iterations Advanced Settings WARNING These parameter settings are for advanced users Shep Size 0 01 Number Steps 10 Max Iterations 1000 Figure 5 54 Advanced Fitting Parameters 88 Autolab SPR KE User manual 5 3 7 Viewing the Analysis Summary The last step of the Analysis Wizard is a summary of the process that was executed The figure below is a screenshot of the summary step for an Integrated Rate Equation analysis The final parameters for each interaction plot are listed as well as the final modelling plot green plots To copy this data to another application use the Copy to Clipboard button r Analysis Wizard Step 7 of 7 BAX Summary Legend Pointer j gt Zoom Plots Region Association Model Monophasic Method Integrated Rate Equation Monophasic Kinetic Parameters ka 9 3 0 4 E4 kd 4 6 2 9 E 4 Advanced Settings Step Size 0 01 Concentration 20nM Start 145 Stop 2411 85 Chi Squared 885 2267 Number of Iterations 28 Parameters 3 Data Points 454 Reduced x 2 1 9628 Parameter Value Error Ks Ks 0 0016 3 7579E 6 E 217 5144 0 1854 0 1733 R 0 11 2275 0 2012 0 7055 E R 0 228 7419 0 2736 lt Mm gt Advanced Fit Details Copy to
89. selecting Projects or from File Open gt Project including Van t Hoff Projects By default the Open file dialog will default to regular Project files with a kpf file extension The user can change this to the kvf file extension as shown in Figure 7 4 to load Van t Hoff Project files Autolab SPR KE User manual assoc_test kpf crop kpt depletion_29000DA kpf Ejer EI Project_temp kpf Project with outliers kpf rebinding kpf Sample6 kpf Sample kpf Figure 7 4 Selecting Van t Hoff Project Files kvf to load in the Open File dialog 7 5 Van t Hoff Equilibrium Analysis The Van t Hoff analysis uses affinity data in the calculation To provide the affinity information each Overlay file must go through an Equilibrium analysis using the Analysis Wizard as discussed in Section 5 3 8 To launch the Analysis Wizard select it from the Activity Bar as shown in Figure 7 5 Analysis oem udlede Wie LONE i Modeling Wizard Signal Processing Van t Hoff Wizard onte Carlo Wizard Figure 7 5 Selecting the Analysis Wizard Autolab SPR KE User manual 117 This action will launch the Analysis Wizard which is discussed in detail in Section 5 3 There is one significant difference between a default Analysis and one in the Van t Hoff project Because the user can import multiple overlay files in a Van t Hoff analysis the first panel of the Analysis wizard will allow the user to sele
90. soft Excel is being used for this example d Press CTRL V on the keyboard or click File gt Edit gt Paste from Excel s Main Menu e The data points from Kinetic Evaluation will now be copied into Excel as shown in the figure below Autolab SPR KE User manual El fie Edit View Insert Format Tools Data Window PEN i aval 0 B z TeS ES Snagit ji window Al be fe Plott Plot KIN1OI _ KIN102 SEL geese ES epee 1 1 5 27255 5 2818 10 1 6881 10 2 5008 15 0 81656 15 1 8964 14 0208 20 1 3752 0 7654 25 0 98722 0 6302 30 0 67913 0 54424 0 45684 0 40355 40 0 27888 0 30382 0 12522 0 23133 50 0 01205 0 16563 55 0 0879 0 0946 60 0 2072 0 04817 0 2722 0 0032 70 0 3471 0 0548 75 0 4137 0 1018 80 0 4302 0 1502 85 0 4766 0 2064 90 0 5558 95 0 2476 95 0 6126 1 0 3059 100 0 7024 105 0 3608 105 0 7575 110 0 4104 110 0 7917 115 0 4556 115 0 8224 5 10 15 20 25 30 35 40 55 60 65 70 75 60 65 90 95 Time3 KIN103 Channel 1 2 3342 0 44214 1 9849 1 41 1 0568 0 84163 0 6031 0 42989 0 29759 0 1483 0 01756 0 1227 0 2343 0 293 0 3765 0 4621 0 62 0 5921 0 6225 0 7469 0 8015 0 642 Ready Fell n ime4 5 10 15 SUM 43 6953 066 KIN104 Channel 1 1 6544 2 fAGT 1 8437 1 2656 0 89699 0 65356 0 4666 0 33069 0 19128 0 06476 0 0444 0 1216 0 2269 0 3172 0 4125 0 5002 0 5774 0 6305 0 6649 0 729
91. ssing Van t Hoff Wizard Monte Carlo Wizard 8 1 Selecting the Monte Carlo Wizard 600 800 Time s 125 E KIN101 5_differential kpf 5 Association L KIN101 5_differential ko Selecting Monte Carlo Wizard will launch the Monte Carlo Wizard Initialize Monte Carlo Number of Runs 100 G Variation Vary the starting parameters of the fit to ensure convergence to the same end result Percent Variation 199 Noise Add noise to the data to estimate the errors from the fit Noise m Legend Pointer MN o gi Response m ah oO oO 600 800 Time s 8 2 The Monte Carlo Wizard 1200 The wizard has the two options discussed above Each of these options will be discussed below in the following sections One feature common to both types of 126 Autolab SPR KE User manual Monte Carlo runs is the number of runs that will be executed for the Monte Carlo The default number of runs for integrated equations is 100 For numerically integrated equations the default is 25 The numerically integrated equations use a lower default as they are more computationally intensive The user can change the default number of runs 8 2 1 Monte Carlo Analysis of Numerically Integrated Fits If the user has selected a Monte Carlo analysis of a fit that uses numerical integration they will see an initial warning Numerical Integration Mont
92. t Files File Type File Description Extension SPR file Spr DA measurement file version 4 2 or greater than 4 2 Exported data from Biacore or CLAMP Within the KE application measurement files are treated as read only files It is not possible to change the contents of a measurement file within the KE application 4 3 1 Opening a Measurement File with Multiple Plots Measurement files that are generated by other applications 1 e Biacore or CLAMP may contain more than one measurement per file If a text file is opened that contains more than one measurement it is up the user to decide how KE will handle the measurements In Figure 4 14 below the user is given the option to open the measurements as separate files i e 1 file per measurement or to directly build a new Overlay see section 4 4 from the measurements in the file File Load Option Multiple Measurements Detected One of the fileja that you are attempting to open contains more than one measurement Please select how wou would like KE to handle this file Load the plots and create a new overlay Load the plots as separate measurements Figure 4 14 Multiple Measurements User Prompt 4 3 2 Creating Sub Curves from a Measurement 42 Autolab SPR KE User manual It is possible to select a section of a measurement and copy it into a new file This type of file is called a KE Sub Curve The following steps show how to create a KE S
93. ta seessseoeenssssssseeeressssssseceressssss 22 Aa VW Orkin With Data sensasine soudialetaducteas ba be a ixetbusdlweadac inns aa 3l g Openin SNCS re REE ED TEE ERE sa satin seine atom diese 31 4 1 1 Opening a File from the Main Menu cc roede 31 4 1 2 Opening a File from the Toolbar s neeeenssssssseeeressssssssssreessssssseceressssss 31 4 1 3 Opening a File from the Activity Bar 20 0 cecccccccccccceesssseeeeeeeeeeeaeeeeees 32 4 2 O WAC WS PINES iets REESE Natsia bee BESES SEERE BESES EEN RE EET FEDE ERE Es SENESTE RENEE SEER 33 421 Merano PIOt VIEW euiar a e acest S 34 ADD Data PO nN CW a E E 35 4 2 3 Event Log History Summary View c cccscccccecceessseeeeeceeeeeeaeeeeees IT AS Meas rement PICS csc or salariilor sne 41 4 3 1 Opening a Measurement File with Multiple Plots sssscseseeerereeeee 4 4 3 2 Creating Sub Curves from a Measurement ccccccsseeeseeeeeeeeeeeeeeeees 4 J 2115 DIEZ BYGD ob cic geen ante ee ee CCN A Se eee 43 4a A Overa y Mando ei cai one atin an er ek Gan SE ele ede nares a 45 B PLOPC CL NCS a E EEOAE 47 AD Creatine a Project Bile an abate hdelatieie 47 ASD 2 Perlormins a ANALYSIS socsseiwccdxd scone beaateaxaincnateelanedadasens De oe enes 48 4 5 3 Navigation of a Project File ccccccccccccssesssseeccccccseeesssesecececeseeeesses 48 aA yes OL Project EB I LE SENERE ESS ESS ESS TES FESSEREE DE
94. that the Synchronize action is selected and the plot to be synchronized is selected in the list of plots Autolab SPR KE User manual 71 The following figure depicts the result of the synchronization Legend k Pointer j Zoom k T Select ha a a CH gt a a wn mm a 2 w a of 800 Time Figure 5 32 Result of the Synchronize action 5 2 8 Zero at Average The Zero at Average action averages the value of the data in the selected region and subtracts this value from the entire plot The user must select a region before using this action as the Full Plot option is not possible The following Figure 5 33 and Figure 5 34 are before and after screenshots of the Zero at Average action Note that the plots have been zoomed into a region to clearly demonstrate the action 72 Autolab SPR KE User manual Legend k Pointer J Zoom rt Select Response m g a mm 1000 4400 12800 Time s Figure 5 33 Zoomed in and Selected Region to Zero Legend k Pointer pp Zoom r 1 Select 400 g0 g0 70 60 50 40 30 os P Pai fi a wn Cc 2 Z uw a o 1000 Time Figure 5 34 Result of the Zero at Average action Autolab SPR KE User manual 7 oS 5 3 Analysis Wizard The Analysis Wizard is used to determine the kinetic binding rates of the interaction plots that are measured with the Autolab SPR Data Acquisition software This section
95. the concentrations of an Association or Dissociation analysis Autolab SPR KE User manual 113 7 Van t Hoff Analysis Wizard 7 1 Introduction Van t Hoff analysis is a technique that links changes in the affinity constant K4 with respect to temperature to the thermodynamic properties AH AS and AC Delta Enthalpy Delta Entropy and Delta Heat Capacity The integrated Van t Hoff equation 1s InK _ AH T AS T AC Z r 7 inf T RT R R T T Equation 7 1 In Equation 7 1 the logarithm of the affinity constant is related to 1 T and the constants AH AS and AC where each constant is evaluated at a reference temperature T typically 25 C Since there are three free parameters at least four measurements of Ka at different temperatures are required to over constrain the three fitted constants This requirement means that the user should measure Reg versus Time for five concentrations at four separate temperatures typically in the range 10 40 C for a total of at least 20 measurements 7 2 Creating a Van t Hoff Project A Van t Hoff Project is created through the New Project builder Select New Project from the Activity bar or File gt New Project Including Van t Hoff Projects from the File Menu These actions will launch the New Project builder as shown in Figure 7 1 Create New Project Import an opened Overlay file Import an O
96. the interaction plot where the analyte is removed from the immobilized ligand Response m in So Determine Concentrations O O Determine the concentrations in the overlay by applying a calibration file 1500 Time s 4 Figure 5 36 The Analysis Wizard The following descriptions correspond to the numbers that appear in Figure 5 36 1 Action Area This region of the Analysis Wizard is where all the user options and configurations take place Each step in the wizard will present a new panel 2 Overlay Data The Analysis Wizard uses the Overlay data from a Project file This graph will display the Overlay plots and eventually will show only the region of the plots that is selected by the user to analyze section 5 3 3 3 Graph Toolbar The figure below depicts the tools that are available for the Analysis Wizard dialog The Plots toolbar option is not available until the end of the Analysis Wizard process see section FI Legend k Pointer gt zoom Plots Analysis Plot Residual Plot Both Plots Figure 5 37 Analysis Wizard Toolbar 4 Navigation Buttons To move step by step through the Analysis Wizard use the Next button To move to a previous step in the wizard 76 Autolab SPR KE User manual use the Back button To quit the wizard at anytime use the Cancel button The following subsections provide a detailed overview of each step in the Analysis Wizard
97. tion These equations are pre integrated before they are used in the fitting routine Due to this pre integration step this model cannot handle complex rate equations The available integrated rate equations are Monophasic and Biphasic It is not possible for the user to create custom integrated rate equations 82 Autolab SPR KE User manual Numerical Modelling This is a more complex method in which the rate equations are integrated numerically during the fitting process The benefit of having the rate equation numerically integrated is that more complex models can be used It is possible for users to construct rate equations to use with the Numerical Modelling method see section 5 1 for more details Select the Analysis Method Integrated Rate Equation Rate equations that are integrated before the fitting process Numerical Modeling Complex rate equations that are resolved by numerncal calculations during the fitting process Figure 5 45 Selecting an Analysis Method 5 3 5 Selecting the Model to Use The next step in the Analysis Wizard is to specify which model to use This step differs depending on which analysis method was selected in previous step see section 5 3 4 Figure 5 46 is a screenshot of the models that are available for the Integrated Rate Equation method The model is specified by selecting it from the drop down list A brief description is displayed for the currently selected
98. tion Jog view Tools Help E G A Pointer 4 Zoom Select E Legend MASS B Measurement1 ksc New Project New Calibration New Van t Hoff Project New Subcurve Measurements 2 Overlays Projects Calibrations Van f Hoff Project a no a o a oO Views A Temperature C View Interaction Plot View SPR Curves View Data Points View Event Log Modeling Wizard Figure 4 18 The newly created Sub Curve 4 4 Overlay Files Overlay files are the result of combing multiple interaction plots Measurement files into one file 44 Autolab SPR KE User manual Note In order to create a new an Overlay file there must be at least one Measurement file opened Overlays are built with the Overlay Builder which is depicted in Figure 4 19 The Overlay Builder can be opened from the Main menu File gt New gt Overlay or by clicking on the New Overlay item in the Activity Bar Select the plots to add to the new overlay Available plots are indicated with a green background Overlay plot names can be edited by double clicki Channel 1 Figure 4 19 The Overlay Builder To create a new overlay select the interaction plots by checking the boxes that you wish to add Items that are present in the measurement are presented with a green background while items with gray background indicate interactions that were not located in the measurement To select all available
99. trations sseeeeeessssssseeeressssssseecressssssseeceeesssssseeeeeees TI 5 3 3 Specifying the Start End Times of the Analysis ccccccsseeseeeeeeeeeeeees 78 5 3 4 Selecting the Analysis Method ccccccccccccseessseeeccccceseeessseseeeeeeenees 81 5 3 5 Selecting the Model to Use M1 W W W ssssnsesveesenesenesssssseneennssessennennsssessenneennee 82 5 3 6 Configuring and Fitting the Model ssnnennessessssseeenssssssssseerssssssssseeeees 84 5 3 7 Viewing the Analysis Summary ccccccccccceeceeseeseeeceeeeeeseeeseeeeeeeeeeeaas 88 Op Oe UNL SS acres een ranen ESKE ERNE ESKE NNE ENKEN KE 91 5 3 9 Equilibrium Analysis Plots at Equilibrium u u ssssseseeeseerrerenrres 91 5 3 10 Equilibrium Analysis Outlier Analysis u u u dsseeseeseeeesereereeeerssenneee 93 5 3 11 Equilibrium Analysis Analysis cccccccccccccsseeseseccceeeeeeeeseeeeeeeeeenaas 95 23912 EGU 1370 DT D S Da 18 2 751 y essien EE EES 97 5 3 13 Determine Concentrations cccccccccccccccccccessssssssssssssssesceccceeeeeesaeenees 98 5 3 14 Setting the Region to Analyze for Determining the Concentrations 99 5 3 15 Determine plots not at equilibrium cc eeeeeeeccecceeeeeeeeeeeeeeeeeeeaaas 99 5 3 16 Apply the Calibration File aa orne eee ERR sr serbere 101 O AU 5784 819 TA SIS eea ERE cases 104 Gi NOW 3 FSA HORSE a EE cies etcennceen
100. ub Curve file a Open a measurement file b Change the graph mode Selection as shown in Figure 4 15 Kinetic Evaluation File View Tools Help mi k Pointer 4 Zoom 1 15 sch Legend ey Figure 4 15 Changing the Graph Mode to Select c Right Click and drag the mouse pointer to select the region that you want to copy into a new measurement file see Figure 4 16 Kinetic Evaluation JER File View Tools Help D ca Gl Rh Pointer 4 Zoom Select Legend MASS IBO Create Files A New Overlay New Project New Calibration New Van t Hoff Project New Subcurve Open Files Measurements 1 Overlays Projects Calibrations Van f Hoff Project Response m Temperature C TR A Pa B et EEN Pha A B ete ree dew WD Fingal PRET LE Bia aA inl to VAP A el NEN can ater LPT Pri SI te FSi are ag oe y View Interaction Plot View SPR Curves View Data Points View Event Log Modeling Wizard Figure 4 16 Selected data to be copied to a sub curve d Once the selection has been made Click the New Subcurve item in the Activity Bar as shown in Figure 4 17 Autolab SPR KE User manual 43 New Overlay New Project New Calibration New Van t Hoff Project New Subcurve Figure 4 17 Choosing the sub curve option e A new file will be created which contains the selected data Figure 4 18 shows the new KE Sub Curve file Kinetic Evalua
101. umber to the right shows the number of opened calibration files Views View Event Log Shows additional information about the file Analysis Signal Processing Launches the Signal Processing dialog Van t Hoff Wizard Launches the Van t Hoff Wizard Figure 2 13 shows the startup state of the Activity Bar In this state the New Overlay action is disabled because a prerequisite is that there must be at least one measurement file open 12 Autolab SPR KE User manual New Overlay New Project New Calibration New Van t Hoff Project New Subcurve Measurements Overlays al Cali Van t Hoff Project Figure 2 13 Startup state of the Activity Bar 2 6 Project Navigation Bar Project files section 4 5 can contain more than one file Since there are multiple files there must be a way to navigate through them When a project file is in focus the navigation bar will be shown on the right of the screen as shown in Figure 2 14 below E Frojectt kpt A BsAB plots kod 5 Association Association Association E quilbriuirn E quilibriuri Figure 2 14 Project Navigation Bar The Navigation Bar provides links to the data within a project These links in the Navigation Bar are grouped into categories The Overlay files will appear directly below the name of the project file For normal projects there is only one overlay For Van t Hoff projects there can be more than one overlay In Figu
102. ve 38 KE Sub Curve file 38 Kinetic Model 50 L Legend 58 Ligand 53 Line Pattern 47 Line Tool 76 Main Menu 2 3 Mathematical Operation 62 Measurement Files 37 Microsoft Excel 34 Modeling Wizard 50 Monophasic 54 77 Monte Carlo Analysis 116 Multiple Plots 38 N Normalize 64 Numerical Modeling 78 O Open Files 10 Opening Files 29 Outliers 89 Overlay Builder 40 Overlay Files 40 P Performing an Analysis 45 Pointer 58 Print Preview 49 Printing 48 Product 53 Project Builder 44 Project Files 44 Project Navigation Bar 2 12 45 Project Types 46 Autolab SPR KE User manual R rate equations 78 Req vs Concentration 91 Residual plot 85 S Select 58 Signal Processing 56 Smooth 65 SPR file 38 start time 75 Summary View 37 Symbol Pattern 47 Synchronize 66 T Text file 38 Toolbar 2 8 Tools Menu 7 133 U User Interface 2 V van t Hoff Analysis Wizard 112 Equation 107 Project 107 Project Builder 108 Saving and Loading projects 109 van t Hoff Analysis 107 Vertical Axis 47 View Menu 5 Viewing Files 31 W Workflow 14 Z Zero at Average 68 Zoom 58 04 2010 Kanaalweg 29 G 3526 KM Utrecht The Netherlands www metrohm autolab com
103. verlay file from Disk Browse Create New Vant Hoff Project Figure 7 1 The New Project builder with the Van t Hoff option selected Select the Create New Van t Hoff Project radio button and click OK This action will launch the Overlay importer 114 Autolab SPR KE User manual 7 3 Importing Overlays The Van t Hoff analysis requires at least four overlays Each overlay corresponds to multiple measurements each taken at different concentrations with each set of overlay measurements taken at the same temperature The Van t Hoff Project Builder is shown in Figure 7 2 The Van t Hoff Project Builder will allow the user to import multiple Overlay files These overlay files will either be already open in KE as shown in the figure or can be imported from disk Van t Hoff Projects are the only projects that allow multiple overlay files to be added to the same project file Select the overlays to add to the project Vant Hoff Analysis requires at least 4 overlays Import an overlay from Disk Use in Vant Hoff Project Overay_temp_1 Overay_temp 2 Overlay temp 3 Overay_temp_4 Overlay temp 5 Figure 7 2 The Van t Hoff Project Builder used to import multiple overlay files to a Van t Hoff Project Once the overlay files are selected the user can import the overlay files into the Van t Hoff project by cl
104. verlay has been added to the Project Any changes to the Overlay in the Project file will not affect the original Overlay file 22 Autolab SPR KE User manual File View Tools Help Zoom i Legend p Properties KIN101 IB0 KIN102 1B0 KIN103 1B0 KIN104 B0 KIN105 1B0 Overlayt kod Project1 kpf Project1 kpf Overlay kod New Overlay New Project New Calibration New Van t Hoff Project Overlays 1 Projects 1 Calibrations Van f Hoff Project View Interaction Plot View Data Points View History Analysis Wizard Modeling Wizard Signal Processing 1500 Time s Figure 3 13 The New Project File 3 5 Using the Analysis Wizard to Fit the Data Now that the Project file has been created we can use the Analysis Wizard to fit the data For this Workflow example we will be performing an Association analysis using the Monophasic Integrated Rate Equation a To launch the Analysis Wizard select the Analysis Wizard item from the Activity Bar as shown in the figure below Autolab SPR KE User manual 23 New Overlay New Project New Calibration New Van t Hoff Project New Subcurve Overlays 1 Projects 1 Calibrations Van f Hoff Project Analysis Wizar Calibration Wiz Conversion Tool Modeling Wizard Signal Processing Vant Hoff Wizard on 1 Lr Monte Carlo Wizard Figure 3 14 Selecting the Analysis Wizard b The Analysis Wizard is a step by step process t
105. ying the Start End Times of the Analysis Step 2 is to set the plot ranges as shown in Figure 6 6 This procedure is the same as procedure for a regular analysis Autolab SPR KE User manual 107 Calibration Wizard Step 2 of 6 CR Set the Region to Analyze Legend amp Pointer zoom E Line KIN102 Channel KIN101 Channel KIN103 Channel KIN104 Channel M O KIN105 Channel Response m Region Options Same Region for All Plots Unique Regions for Each Plot Show In dR dt vs t 1500 Time s Figure 6 6 Setting the plot ranges for a Calibration project 6 5 Extrapolation to Equilibrium Step 3 as shown in Figure 6 7 determines any plots that are not at equilibrium by fitting the data The fit is accomplished by pressing the Fit button Any plots not at equilibrium will be checked in the Not at Equilibrium column The fit extrapolations are shown in the plot area 108 Autolab SPR KE User manual Calibration Wizard Step 3 of 6 BAX Set Plots Not At Equilibrium Legend k Pointer gt Zoom Max Max from Data Fitting gt KIN102 Channel 1 191 61 192 56 KIN101 Channel 257 11 258 49 KIN103 Channel1 322 33 323 35 KIN104Channel1 337 23 337 27 KIN105 Channel1 358 23 357 42 Threshold to consider curve at equilibrium o az c a no vw oe 1500 2000 2500 3000 Time s Figure 6 7 Equilibrium extr

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