Page 1 GE Healthcare Biacore T100 Software Handbook...
Page 3 4.2.1 Injection sequence ................... 32 4.2.2 Assay setup ....................34 4.2.3 Injection parameters................35 4.2.4 Sample and control sample tables ........... 36 4.2.5 System preparations................37 4.2.6 Rack positions .................... 39 4.2.7 Prepare Run protocol................43 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 4 5.9.4 System preparations................99 5.9.5 Rack positions ..................100 5.9.6 Prepare Run Protocol................100 5.9.7 Starting the run ..................100 5.10 Requirements for assay-specific evaluation ......100 5.10.1 Concentration analysis ................100 5.10.2 Kinetics/Affinity..................101 5.10.3 Thermodynamics..................101 5.10.4 Affinity in solution...................101 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 5 7.2.5 Adjusting plots for controls ..............130 7.2.6 Ranking .......................132 Bar chart items ................133 7.3.1 Selector functions ...................133 7.3.2 Display options ..................134 Report point table ..............134 7.4.1 Displaying the report point table .............134 Concentration analysis Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 6 9.8.5 Kinetics – Two State Reaction............186 9.8.6 Affinity – Steady State 1:1 ..............188 Creating and editing models ............188 9.9.1 Interaction models for kinetics ............189 9.9.2 Equation models for kinetics .............194 9.9.3 Models for steady state affinity ............195 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 7 B.4 Inject and Recover ..............221 B.5 Kinetics heterogeneous analyte ..........223 B.6 L1 liposome capture ..............223 B.7 LMW kinetics and LMW Screen ..........224 B.8 NTA kinetics ................. 226 B.9 Single-cycle kinetics ..............227 Index....................229 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 8 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 9: System Overview
This Handbook describes in detail how to use the Control and Evaluation Software. System overview Instrumentation in the Biacore T100 system is described in full in the Biacore T100 Instrument Handbook. Important features relevant to software operation include: •...
Page 10: Support For Use In Regulated Environments
) environments is provided in an optional package that adds appropriate functionality to the Biacore T100 software. Functions for GxP support are described in a separate Biacore T100 GxP Handbook. Descriptions of software in the current Handbook apply to installations both with and without the GxP package unless otherwise stated.
Page 11 The response may be absolute (above a fixed zero level determined by the detector) or relative to the response at another specified report point. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 12 1 Introduction 1.4 Biacore terminology Figure 1-2. Schematic illustration of a sensorgram. The bars below the sensorgram curve indicate the solutions that pass over the sensor surface. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 13 Control Software Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 14 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 15: Operational Modes
Control Software – general features 2 Control Software – general features Operational modes Biacore T100 Control Software offers three modes of operation: • Manual run provides interactive control of the instrument operation, executing commands singly as they are issued. This mode is most useful for ad hoc experiments involving one or a few injections, such as testing the response obtained from injection of a single sample.
Page 16: User Interface
The content of the status bar varies between different situations: for wizard- and method-based runs, the elapsed run time and the estimated total run time are included. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 17: On-Line Help
Export exports the current results to a file in Microsoft Excel or XML format, or exports the contents of the report point table to a tab-separated file. See Appendix A for details of the export format. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 18 When you close the software with Exit while the instrument is still switched on, you may choose to shut down the instrument for a shorter or longer period if required. See the Biacore T100 Instrument Handbook or the on-line help for more details.
Page 19: Edit Menu
[Blank] for flow cells that have been prepared as a blank reference surface by activation and deactivation. The text [Incomplete results] indicates that the immobilization run was interrupted (by for instance user intervention or power failure) before it could be completed. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 20 When the reference line is displayed, choosing Baseline sets a baseline at the current reference line position, and the coordinates window shows the response relative to that baseline. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 21: Run Menu
Standby puts the instrument in standby mode, which maintains a low buffer or water flow through the flow system for up to 4 days. Leaving the instrument in standby mode when not in use is generally recommended. The instrument is Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 22: Right-Click Menus
Insert Chip and Eject Chip are used for docking and undocking the sensor chip respectively. More details are given in Chapter 3 of the Biacore T100 Instrument Handbook. Set Temperature sets the sample compartment and analysis temperature.
Page 23: File Storage
Results are saved in files with the file name extension .blr. Result files from wizard- or method-based runs contain a copy of the wizard template or method as well as the results of the run. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 24 2 Control Software – general features 2.4 File storage Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 25: Preparing For A Manual Run
You insert the samples as part of the starting procedure for the run. You can also add samples during the run. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 26: Starting A Manual Run
Click Eject Rack to eject the rack tray so that you can load your samples. Click Start to start the run. You will be asked to specify a result file name before the run actually starts. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 27: Controlling A Manual Run
You can also use the right-click menu to copy selected command or commands and paste them elsewhere in the queue. The Copy function works with both completed and pending commands. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 28 If you want to place the command later in the queue, use the right-click menu in the queue panel to insert the command at the appropriate place. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 29: Ending A Manual Run
Stop Run command in the appropriate position. Choose Tools:Eject Rack to eject the rack tray and remove your samples and reagents. Choose Tools:Eject Chip to undock the chip if desired. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 30 3 Manual run 3.4 Ending a manual run Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 31: Wizard Templates
Application wizards Application wizards guide you through the procedure of setting up common applications, with recommendations and settings based on GE Healthcare’s expertise in the field of SPR-based interaction studies. Wizards are an ideal starting point for inexperienced or infrequent users, since they offer a structured sequence of settings that covers all essential aspects of the assay in question.
Page 32: Running Wizards
4.2.1 Injection sequence This dialog determines the sequence of injections in the wizard analysis cycle. Some injections are not supported in certain wizards (e.g. the kinetics wizard does not support enhancement injections). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 33 This is the sample to be analyzed. The solution used for the sample injection is normally different in different cycles, and is specified in the sample table at a later stage in the wizard. The sample injection is required in all wizards. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 34: Assay Setup
For these chips (Sensor Chip C1, CM-series and SA) the option to run conditioning cycles is unchecked by default. You may want to use conditioning cycles in cases where Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 35: Injection Parameters
The Injection parameters dialog specifies details of the injections selected in the Injection sequence. Details of this dialog box may vary according to the injections selected and the particular wizard. Some features may be generalized: Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 36: Sample And Control Sample Tables
Sample and Control Samples steps respectively. The details of these steps differs according to the wizard purpose, but the following general features may be noted. Further details are given in the respective wizard descriptions later in this chapter. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 37: System Preparations
Sample details can be imported from an external file if this option is enabled under Tools:Preferences. See Appendix A for details of import formats and procedures. 4.2.5 System preparations This dialog box specifies how the system will be prepared before the first cycle. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 38 Note: Both analysis temperature and sample compartment temperature can be set in advance with the Set Temperature option from the main Tools menu (Section 2.3.6), to allow the temperature to equilibrate before setting up the assay wizard. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 39: Rack Positions
This dialog box shows where samples and reagents are placed in the microplate and/or rack. Positions are color-coded by region according to sample and reagent categories: you can change the color-coding in the Automatic positioning dialog, accessed through the Menu button. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 40 (Ctrl-V) in another. Positions can also be reorganized using the Automatic positioning dialog (see below). Menu functions Use the Menu button to access additional functions for rack positioning. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 41 This option controls whether the samples and reagents will be placed in the reagent rack or the sample microplate. If Auto is chosen, placement is decided on the basis of number and volume of solutions in the region. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 42 Appendix A for details of the exported file format. Print Rack Positions Prints a copy of the rack positions diagram and table. Print Wizard Template/Print Method Prints a copy of the currently open wizard template or method. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 43: Prepare Run Protocol
Buffer requirements for standby after the run (approximately 65 ml/day) are not included. The Menu button provides options for saving and printing the wizard template (Section 4.2.6). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 44: Surface Preparation Wizards
Enter the buffers and pH values to be used for scouting. The default list covers sodium acetate buffers in the pH range 4 to 5.5, available as ready-to-use solutions from GE Healthcare. Buffers will be tested in the order listed. Note: The buffers listed here are buffers in which the ligand should be prepared.
Page 45 The same overlay plot is created when a saved run from Immobilization pH Scouting is opened in the Evaluation Software. Note: Opening a saved run in the Control Software does not generate the overlay plot automatically. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 46: Immobilization
See the Biacore Sensor Surface Handbook for more information about ligand immobilization. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 47 Choose the immobilization method. Predefined methods are provided for standard immobilization chemistries. Customized methods can be defined by clicking on the Custom Methods button (see below). Predefined methods are marked with a T100 icon ( ) in the selection lists. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 48 150 µl is reached. This option can be used to conserve valuable ligand without losing the benefits of aiming for a target immobilization level, and can be useful for sensor chips where preconcentration cannot be performed, e.g. Sensor Chip Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 49 The ligand injection is created automatically and cannot be deleted: solution and contact time for the ligand injection are specified in the main wizard dialog. A method may only contain one ligand injection. Other injections have the following functions: Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 50 Step 2. System preparations Check the System preparations options as required (see Section 4.2.5). Step 3. Rack positions Accept or change the rack positions for the various solutions required (see Section 4.2.6). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 51 EDC/NHS. For low ligand levels, this value usually gives a better indication of the amount of ligand immobilized. Result files from immobilization can also be opened in the Evaluation Software if you want to prepare other sensorgram displays or plots (see Chapter 7). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 52: Assay Development Wizards
Regeneration scouting always includes one startup cycle with the same injection sequence as the scouting cycles but with injection of buffer for all injections. Note that this differs from the startup cycle construction in the other application wizards. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 53 Check High viscosity solution if any of the regeneration solutions tested has a relative viscosity higher than about 3 (corresponding to about 35% glycerol or 40% ethylene glycol at 20°C). This will modify the injection procedure for better handling of viscous solutions. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 54 Note: Report points are set before the sample injection for baseline and shortly after the sample injection for sample response. Thus the points for the first cycle indicate the starting values, while those for subsequent cycles each indicate the effect of the previous cycle. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 55: Buffer Scouting
(in which case a single flow cell will be used for each buffer, with flow cells 1, 2, 3 and 4 for buffers A, B, C and D respectively). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 56 Ligands for capture, samples and enhancement reagents should be prepared in each of the buffers tested. Separate rack positions will be created for samples in each buffer (for example, running buffer scouting with 4 buffers and 5 samples will require 20 sample positions). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 57 When the wizard run is completed, the results are opened automatically in the Evaluation Software. In addition to the general predefined plots (Section 6.4), plots of binding and stability against sample are created to visualize the behavior in the different buffers. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 58: Surface Performance
Step 3. Injection parameters Specify the injection parameters for each injection in the cycle (see Section 4.2.3). Step 4. System preparations Check the System preparations options as required (see Section 4.2.5). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 59: Assay Wizards
Specify the conditioning and start-up cycles (see Section 4.2.2). Step 3. Injection parameters Specify the injection parameters for each injection in the cycle (see Section 4.2.3). One sample injection panel will be created for each sample Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 60 Click Import to import the sample data from an external file. Import of sample information must be enabled in Tools:Preferences to use this function. See Appendix A for details of import functions and file formats. Click Control Samples to enter control samples for the run. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 61 Binding analysis results When the wizard run is completed, the results are opened automatically in the Evaluation Software. Predefined plots (Section 6.4) are created for each sample. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 62: Concentration Analysis
The value specified for Fraction refers to the proportion of the fixed component in the final mixture: for example, a value of 30% will mix 7 parts of sample with 3 parts of the specified solution. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 63 To run replicate concentrations, enter the same concentration on multiple rows. Calibration points will be run in the order entered. You can choose a different concentration unit if required from the pull- down list in the table header. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 64 Control samples are specified in terms of sample ID and expected concentration. The expected concentrations should lie within the range covered by the calibration curve. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 65 Save the wizard template if required and start the run. Evaluation of concentration assays Chapter 8 describes how to evaluate concentration assays. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 66: Kinetics/Affinity
30-second injection at 40 µl/minute, regardless of other settings in the wizard. Report points are set automatically for the carry-over injection to allow evaluation of potential carry-over problems. Step 2. Setup Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 67 Section 4.2.3). The sample injection for kinetics measurement has an additional setting for dissociation time. This is the time for which dissociation will be monitored after the end of the injection without disturbances from flow system washing procedures. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 68 A, B, A, A, B, A, B, B… will be run in the order A, A, A, A, B, B, B, B… The Run order setting applies within each concentration series. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 69 Save the wizard template if required and start the run. Evaluation of kinetics/affinity assays Chapter 9 describes how to evaluate kinetics/affinity assays. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 70: Thermodynamics
Specify the conditioning and start-up cycles (see Section 4.2.2). Start-up cycles are recommended for thermodynamics experiments to ensure that the response drift that may occur with a new chip or when the temperature is Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 71 Enter the details of the samples. This dialog box is equivalent to the concentration series dialog for kinetics determination (see Section 4.5.3, step 4). Separate rack positions will be created for each sample at each temperature. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 72: Control Experiment Wizards
The control experiment wizards have the same basic structure as the kinetic analysis wizard, but some of the analysis settings are fixed. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 73: Mass Transfer Control
Note: The Injection Parameters step allows you to enter a stabilization time after each sample injection, but all other settings in this dialog are fixed in the wizard. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 74: Evaluation Of Control Experiments
This example shows a clear dependence of binding rate on flow rate, indicating mass transfer limitations in the observed binding. It may however still be possible to extract kinetic information from the sensorgrams. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 75 This example shows a clear dependence of dissociation rate on contact time, indicating linked reactions in the interaction model. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 76 4 Application wizards 4.6 Control experiment wizards Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 77: Opening Methods
Methods 5 Methods Methods in Biacore T100 offer flexibility in instrument control, providing support for applications that cannot conveniently be handled with wizards. Methods are constructed with the Method Builder tool as described in this chapter. Templates from application wizards can be opened in Method Builder (Section 5.1) to provide a starting point for development of customized applications.
Page 78: Method Structure
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Page 79: Method Overview
+- marking or use the Expand All button to expand all commands in the panel. This screen is for information only: settings cannot be changed here. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 80: General Settings
Check the Vary with analysis temperature box to set the sample compartment temperature automatically to the same value as the analysis temperature. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 81: Assay Steps
Recurrence. To create a new assay step, click New Step. The step will be created with default settings at the end of the current method. Move the step in the method with the Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 82: Base Settings
Assay steps are assigned a purpose, used to identify cycles in the evaluation software. The choice of purpose can help to document the method structure, and also determines the way the data is treated in evaluation. Choose the purpose from the list. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 83 Sample step. Undefined Used for assay steps that do not fit the predefined purposes. Assay steps with Undefined purpose will not be included in application-specific evaluation. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 84: Number Of Replicates
If the higher level step is run in replicate, a recurring step is distributed among the total number of cycles including replicates. This is illustrated by the calculations below for a recurring step set to Every 5 cycles: Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 85: Assay Step Preparations
Select the running buffer to be used for the assay step. The default buffer is A (corresponding to buffer bottle and tubing A on the instrument). Cycle types Cycle types define the detailed sequence of operations to be performed in each assay step. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 86: Commands
) buttons to change the position of the selected command in the cycle, and the Remove button to remove the selected command from the cycle definition. Commands are executed from top to bottom in the cycle definition. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 87 The illustration below shows a cycle which will perform an additional regeneration if the relative response after the first regeneration exceeds a specified value: Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 88 If…then construction. You can use the Move up and Move down buttons to rearrange the order of commands within a branch, but you cannot move commands outside the branch in which they are placed. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 89 Biacore analysis with mass spectrometry (MS). Most of the parameters for the command can be set as variables. This command can only be used for injection over all four flow cells: normally, the same ligand should be immobilized in each cell. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 90 The same target position is used for recovered analyte from all repetitions. Note that only one aliquot of deposition solution is used, regardless of the number of repetitions. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 91 Notes: Methods that include the InjectAndRecover command require a sample and reagent rack and cannot be used with microplates (see the Biacore T100 Instrument Handbook for rack details). The contact time for sample, flow rate and number of repetitions determine the total injected volume for both sample and recovery solution.
Page 92: Variables
(for example, kinetic evaluation requires a variable called Conc which holds the analyte concentration). These are selected from a predefined list. Other evaluation variables may be freely defined by the user, to hold Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 93 Analyte molecular weight: required for molecular weight adjustment of report points, and for kinetic evaluation when the concentration is entered in weight-based units. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 94 (the concentration of B is kept constant) ConcA-variable Concentration of interactant A in the sample mixture (the concentration of A is varied) *See Section 11.1.1 for details of how this assay is set up. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 95: Report Points
A window of 5 seconds is adequate for most purposes. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 96: Variable Settings
Columns in the table correspond to variables for the cycle type used in the assay step, and are grouped according to commands in the cycle type definition. Use the right Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 97: Verification
5.9.1 Detection Set the flow path for the method in the Detection dialog. You can only choose a flow path that is consistent with the Detection setting for the method (see Section 5.4). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 98: Variables
Method variable values must be entered in all tables before you can continue to the next step. Evaluation variables may be left blank if desired at this step and values entered in the Evaluation Software. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 99: Cycle Run List
Check through the cycle list to confirm that the variable tables are correctly filled in. Click Overview to display the method overview (Section 5.3). 5.9.4 System preparations Choose which preparation steps should be executed before the method starts. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 100: Rack Positions
Conc. At least two different concentrations are required for linear calibration curves and at least four for 4-parameter fitting. • Samples in the Sample step will normally not have specified concentrations. If concentrations are specified, they will be ignored. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 101: Kinetics/Affinity
At least one assay step is required with purpose Calibration and one with purpose Sample. Both assay steps must be connected to a cycle type that includes one Sample command. The two assay steps will normally be connected to the same cycle type. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 102: Other Requirements
Other requirements Application of solvent correction (see Section 6.7) requires an assay step with purpose Solvent Correction, connected to a cycle type that includes at least four Solvent Correction commands for different solvent concentrations. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 103 Evaluation Software Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 104 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 105: User Interface
This chapter describes the organization of the evaluation software. The various evaluation functions are described in detail in the following chapters. User interface 6.1.1 Organization The Biacore T100 Evaluation Software screen is divided into three main regions: Menu and toolbar Evaluation explorer Work area Status bar •...
Page 106: The Evaluation Explorer
To open multiple result files in the same session, use the File:Append file option. You can only append result files, not evaluation sessions. Appending a file to a session will delete all the user-defined evaluation items in the session. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 107: Common Display Functions
Displays the sensorgram relating to a point in the plot. This option is only available when you right-click on a point: the sensorgram is displayed in a separate window that must be closed before you can continue with the evaluation. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 108 Exports the curves in the current display to a tab-separated text file, for import to third-party software. Complete data is exported regardless of the scale setting of the screen display. Unzoom Restores the previous zoom setting. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 109: Predefined Evaluation Items
Binding to reference Relative response for report point stability against cycle number for the reference flow cell. Capture Relative response for report point capture_level against cycle number for the capture injection. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 110: Custom Report Points
Control Software (Section 2.3.3), are not listed in the Custom Report Points dialog and cannot be edited or deleted in the Evaluation Software. Choose Tools:Custom Report Points to add and edit custom report points. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 111: Adding Report Points
Click Add to add a new report point. Enter the id for the report point (maximum 30 characters) in the Id field. The report point id must be unique within an evaluation session. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 112: Editing And Deleting Report Points
If you delete a custom report point that is used as baseline for other report points, the relative response can no longer be calculated for the latter points. You will be warned if this situation arises. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 113: Keywords
Click in the filter row (directly below the column header) and select a value to display only rows with that value for the chosen column. Click Reset All Filters to restore all filters to the [All] setting. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 114 Note: All user-defined evaluation items are deleted from the session when changes are made to the keyword table, since the changes may affect existing evaluation items. To avoid losing work, save your evaluation session before editing or deleting custom report points. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 115: Solvent Correction
Such variations are unavoidable in the preparation of diverse samples such as drug candidates for screening applications. The solvent correction procedure corrects for the variations arising from the excluded volume effect in these cases. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 116: When Solvent Correction Should Be Used
–150 RU in the reference sensorgram corresponds to a solvent error of +5 RU in the reference-subtracted sensorgram. 3. The reference-subtracted sensorgram is corrected by subtracting the solvent error. This procedure is applied to every point during sample injection. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 117: Applying Solvent Correction
Statistical fitting parameters (chi- squared) are shown for each correction curve in the cycles list. Right-click on a point or curve in the right panel and choose Show sensorgrams if you want to Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 118 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 119: Evaluation Methods
Click Skip to create the method with no kinetics/affinity item included. Kinetic/affinity items that use data sets with multiple R cannot be saved in evaluation methods. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 120: Applying Evaluation Methods
Click Apply to apply the method. Items in the method will be created where possible. Items that cannot be created will be reported in a dialog box. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 121: Sensorgram Items
Hold the cursor over a sensorgram to display a tool tip identifying that particular curve. The sensorgram coloring can be changed if desired with the Tools:Color by option. The following sections describe display functions specific to sensorgram items. General display functions are described in Section 6.3. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 122: Selecting Sensorgrams For Display
This function can be useful for removing injections with high bulk contributions (such as regeneration injections) or other visual disturbances from the display. Choose Undo Cut from the right-click menu to restore the removed data. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 123: Sensorgram Adjustment
(e.g. adjusting analyte response for varying capture levels, by adjusting the baseline to 0 and the capture level to 100). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 124: Markers
Report points and Event markers options respectively under the Tools button. Report points are displayed on the curve and event markers on the x-axis. Figure 7-1. Sensorgram display with labels and markers shown for both report points and events. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 125: Plot Items
Tool tips identify the data points (place the cursor on a point for a couple of seconds to display the tool tip). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 126: Selector Functions
Selection operates in the same way as in the sensorgram window (Section 7.1.1). Other general display functions are described in Section 6.3. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 127: Table Functions
You can also change the order in which columns will be displayed using the Move up and Move down buttons (the top of the column list represents the left-hand column in the table). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 128: Sorting The Plot
The numerical fitting results are displayed in an extra panel below the plot. Choose Curve Fitting:Curve parameters under the Tools button to toggle display of this extra panel. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 129 The closeness of fit is reported for linear fitting as the correlation coefficient R and for 4-parameter fitting as the chi-squared value (see Section 9.7.1). Curve fitting cannot be applied to plots that have been sorted. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 130: Adjusting Plots For Controls
If no sample is chosen as a negative control, 0 RU is used as the negative control response. • The fitted line(s) are transformed to straight horizontal lines with values 100 for the positive control and 0 for the negative control. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 131 (Figure 7-2). Figure 7-2. Polynomial function fitted to 3 control points (highlighted) with the resultant adjustment for controls. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 132: Ranking
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Page 133: Bar Chart Items
This selection applies to all report points shown in the chart. Points for which the selection is invalid are not shown (for example, the report point baseline will normally not be shown on a bar chart of relative response). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 134: Display Options
The report point table is created automatically as an evaluation item. Each evaluation session can only have one report point table item: the table is updated automatically if you add custom report points or apply solvent correction. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 135 Report point time in seconds from the start of the cycle. Window (s) Report point time window in seconds. AbsResp (RU) Absolute response in RU, calculated as the mean value over the time window. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 136 The report point table can be sorted by any column in ascending or descending order for any column. Click in the header for a column to sort the table by that column. Each click in the same header toggles the sort order. Header row Filter row Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 137 Choose Copy Table to copy the whole table. The contents will be copied in tab- separated text format to the Windows clipboard, and can be pasted from there into other programs. All selected cells will be copied, including header cells and filter settings. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 138 7 Data presentation tools 7.4 Report point table Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 139: Requirements For Concentration Evaluation
Concentration analysis 8 Concentration analysis Biacore T100 software supports two approaches to concentration analysis: • Using a calibration curve determined by measurement of known samples (referred to as calibrated measurements). • Calibration-free measurements that rely on the relationship between the analyte concentration and the rate of mass transfer of analyte to the sensor surface.
Page 140: Evaluating Calibrated Concentration Analyses
Settings on the Calibration tab define the report point and fitting function that are used to create the calibration curve. The settings apply to all calibration curves in the evaluation. Choose the settings appropriate for your analysis: Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 141 Changing sample names in calibration cycles only changes the title in the calibration curve panel and has no effect on the curve itself. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 142: Control Samples
Right-click on a sample row in the table or a sample point in the plot and choose Exclude Cycle to exclude that sample from the sample evaluation. Excluded cycles are shown as open symbols and are marked in the table with red strikethrough text. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 143: Samples
Right-click on a sample row in the table or a sample point on the curve and choose Exclude Cycle to exclude that sample from the average sample calculation. Excluded cycles are shown as open symbols and are marked in the table with red strikethrough text. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 144: Custom Models For Calibration Curves
It is for example in principle possible to append a kinetic analysis result file to a concentration analysis, and then apply concentration analysis evaluation: Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 145: Evaluating Calibration-Free Measurements
To evaluate calibration-free concentration analysis, choose Concentration Analysis:Calibration-free from the evaluation toolbar, or choose Add Concentration Analysis:Calibration-free from the Evaluation menu. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 146: Selecting Samples
The option Use reference subtracted data is checked by default if a reference surface is included in the run setup. You should generally perform the evaluation using reference-subtracted data, but you can uncheck this option to use unsubtracted data if you wish. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 147 (so the binding rates should in theory be equal and the QC ratio should be 0). Samples where the QC ratio is negative do not show sufficient mass transport limitation for calibration-free concentration measurements. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 148 Click Included blanks to display an overlay plot of blank cycles in the run. Remove the check-mark from the Include column to exclude a blank cycle from the evaluation (for example if the sensorgram shows unreasonable response levels or unacceptable disturbances). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 149: Removing Disturbed Data
When you are satisfied with the data selection, click Next to start the evaluation. Results appear in the table as each sample series is completed. The evaluation progress is shown in the sensorgram panel. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 150: Interpreting The Results
Samples where the QC ratio of the i is less than about 0.13 should be discarded (see Section 8.3.1). Results for samples where the initial binding rate at 5 µl/min is outside the range 0.3–15 RU/s should be treated with caution. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 151: Fitting Model
20°C. The value at 20°C is provided as an evaluation variable when the assay is run (Section B.2). Parameters for calculating the mass transport coefficient from the diffusion coefficient are fixed in the software. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 152 8 Concentration analysis 8.3 Evaluating calibration-free measurements Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 153 Kinetics and affinity analysis 9 Kinetics and affinity analysis Biacore T100 offers three main functions for analysis of interaction kinetics and affinity: • Kinetics and affinity measurements on the sensor surface, which determine the interaction characteristics between ligand and analyte.
Page 154: Requirements For Kinetics And Affinity Evaluation
• for multi-cycle kinetics, duplicate determinations for at least one non-zero concentration. These conditions are recommended but not mandatory in the Kinetics/Affinity wizard. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 155: Evaluating Kinetics And Affinity In Single Mode
Ligand name. Multiple choices may be available if the ligand is captured and varies between cycles. Result files from versions of Biacore T100 Software earlier than 2.0 do not contain a keyword for Ligand name, and the name will be shown as N/A.
Page 156 The adjustment compensates for small systematic discrepancies in the interval between the start of the injection as recorded in the event log and the time that the Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 157 Click Next> when you have selected the curves to be evaluated. The second dialog shows the blank subtracted curve set and allows you to delete selected regions from all or selected curves, for example to eliminate spikes or other disturbances. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 158 The top panel shows the plot of R against C, based on average response values over the region marked on the sensorgrams. Click Settings to adjust the region used for calculation of R values. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 159 Clicking on Accept will stop the Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 160 The quality control criteria are discussed in Section 9.4.1. This tab is only shown for kinetic evaluations using the predefined 1:1 model. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 161 Report tab are defined in the model. Global parameters are listed on a single row at the top of the table, and local parameters are listed on one row for each curve. • Parameters shows the values for all parameters in the fitting equations. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 162: Multiple Ligand Densities
The same sample name and analysis temperature apply to all subsets. Click on a subset to manage the contents of the subset. You cannot mix subsets that use different samples or different temperatures. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 163: Batch Mode Evaluation
To perform evaluation in batch mode, start the kinetics/affinity evaluation (Section 9.2.1) and check the Batch mode option in the first dialog. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 164: Quality Assessment For Kinetics Evaluation
1:1 model only) gives a brief overview of the reliability of the results. If you prefer, you can hide the quality control tab by setting the appropriate option in Tools:Preferences on the main menu. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 165 Fail: quality assessment unacceptable (Blue) Neutral or user assessment required The quality of the fitting is assessed in five areas: Magnitude of kinetic constants Biacore T100 can reasonably measure kinetic constants in the range 10 –10 for k and 10 – 0.5 s for k .
Page 166 Bottom right: Slow association and dissociation, giving insufficient curvature in both association and dissociation phases. Evaluation is possible but will not be very reliable. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 167 On the other hand, Fail status in any of the parameters is a reliable warning indicator. Base your assessment on the overall quality of the results and the fitting, taking all quality control parameters into account. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 168: Statistical Parameters
For ease of comparison between parameters with widely different absolute values (e.g. k and k ), the standard error may be expressed as a T-value, which is obtained by dividing the value of the parameter by the standard error. A high Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 169 U-values above about 25 indicate that absolute values for two or more of the parameters (rate constants and R ) are correlated and cannot be determined. If the U-value is below about 15 the parameter values are not significantly correlated. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 170: Components Of The Fit
This tool is only available for results obtained with the 1:1 fitting model. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 171 The tool display allows the simulated difference curves to be compared to the experimental residuals Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 172: Quality Assessment For Affinity Evaluation
To help in this assessment, the calculated K value is indicated as a vertical line at the corresponding analyte concentration. The line is red and broken if the value is greater than half the highest concentration. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 173: Summarizing Kinetics And Affinity Results
The summarized kinetic and affinity results are presented as thumbnail plots of the kinetics and evaluation items and as a table of result data. Multiple fits in the same item in the Evaluation Software are presented as separate fits in the summary. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 174 Extended Shows more detailed identification of each thumbnail than the standard view. Thumbnail display settings Choose Display Settings from the View menu or the right-click menu for display settings for the thumbnails. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 175 (Copying a graph and a thumbnail is equivalent when small thumbnails are displayed, since there is no additional information in this mode.) Copying all items copies all the included items in the summary (regardless of Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 176: On-Off Rate Maps
Click on a point in the on-off rate map or select a row in the table below the map to display the thumbnail for the evaluation. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 177: Curve Fitting Principles
The fitting process begins with initial values for the parameters in the equation set, and optimizes the parameter values according to an algorithm that minimizes the chi-squared value (Section 9.4.2) for the fitting. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 178: Local And Global Parameters
It is however justified to use a local R if there is reason to believe that the ligand activity may vary between cycles (e.g. in a capture assay, if the capture level varies between cycles). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 179: Predefined Models
(m h, w, l are the flow cell dimensions (height, width, length in m) One form used in fitting models in Biacore T100 is referred to as the mass ⋅m⋅s transfer constant k (units RU⋅M...
Page 180: Kinetics – 1:1 Binding
Analyte binding capacity of the surface (RU) Rmax Conc Analyte concentration (M) Conc Flow rate-independent component of the mass transfer constant Flow Flow rate (µl/min) tc × f Mass transfer constant Bulk refractive index contribution in the sample Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 181: Kinetics – Bivalent Analyte
Flow rate (µl/min) Provided as input Sample injection start time (s) Provided as input tOff Sample injection end time (s) Provided as input Bulk refractive index contribution in the sample Fitted Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 182: Kinetics – Heterogeneous Analyte
The model cannot evaluate interactions where the proportions and relative sizes of the analytes are unknown. A1 + B = A1B A2 + B = A2B Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 183 (Rmax1/Rmax2) / (MW1/MW2). Flow rate (µl/min) Provided as input Sample injection start time (s) Provided as input tOff Sample injection end time (s) Provided as input Bulk refractive index contribution in the Fitted sample Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 184: Kinetics – Heterogeneous Ligand
In cases where the heterogeneous ligand model is found to give the best fit to the observed sensorgrams, further experimental efforts to reduce the heterogeneity are recommended where possible. A + B1 = AB1 A + B2 = AB2 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 185 Association rate constant for the first and second ligands (M Dissociation rate constant for the first and second ligands (s Equilibrium dissociation constants (M) kd1/ka1 kd2/ka2 Rmax1 Analyte binding capacity of the first ligand Rmax1 (RU) Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 186: Kinetics – Two State Reaction
Reverse rate constant for the conformational Fitted change (s Rmax Analyte binding capacity of the surface (RU) Fitted Conc Analyte concentration (M) Provided as input Flow rate-independent component of the Fitted mass transfer constant Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 187 Analyte binding capacity of the surface (RU) Rmax Conc Analyte concentration (M) Conc Flow rate-independent component of the mass transfer constant Flow Flow rate (µl/min) tc × f1/3 Mass transfer constant Bulk refractive index contribution in the sample Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 188: Affinity – Steady State 1:1
Note: Custom models defined in software versions earlier than 2.0 can be imported and viewed but cannot be edited.These models do not support evaluation of single-cycle kinetics. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 189: Interaction Models For Kinetics
Note: Numbers are used as part of the identifier, not in the conventional chemical sense of stoichiometry. Thus a complex named AB2 does not imply two molecules of B binding to one of A. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 190 Check the box and enter the molecular weight parameter for the analyte to which the binding capacity parameter refers. Binding capacity for the other analyte will be calculated using the molecular weight values. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 191 Enter parameter names for the forward and backward rate constants for each line in the reaction scheme. (The terms k-forward and k-backward apply to the reaction as entered in the scheme, reading from Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 192 In the Report panel, define the parameters you want to appear in the Report tab of the results. Report parameters are defined by a name that may be chosen freely and a value that is entered as a parameter or expression containing parameters. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 193 You can select the equations in the display and click Copy to copy the equations to the Windows clipboard. Use this function and paste the equations in to e.g. Wordpad to print a copy of the rate equations. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 194: Equation Models For Kinetics
Select Equation model in the subsequent dialog. Parameters and report parameters are defined in the same way as for kinetic models. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 195: Models For Steady State Affinity
Note: Beware of trying to define and use complex models for steady state affinity. Because of the relatively few points available for fitting to steady state affinity models (typically about five concentrations in duplicate), complex models tend to give unstable fitting behavior. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 196 9 Kinetics and affinity analysis 9.9 Creating and editing models Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 197: Background
Thermodynamic analysis 10 10 Thermodynamic analysis Biacore T100 supports automated measurement of kinetics or affinity at a series of temperatures using the Thermodynamics wizard (Section 4.5.4). In addition to displaying the variation of kinetic and affinity constants with temperature, the evaluation software extracts standard thermodynamic parameters from the data.
Page 198: Transition State Thermodynamics
Note that the Eyring equation does not have a corresponding non-linear form that takes account of the heat capacity change for transition state formation. Non-linear fitting to obtain values for ΔC ° can only be applied to equilibrium thermodynamic analysis. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 199: Performing Thermodynamic Analysis
In some cases it may be necessary to examine the kinetic or affinity evaluation items to determine which constants belong together. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 200 Click Next> to display the van’t Hoff and Eyring plots together with a table of thermodynamic constants for the equilibrium and transition state formation. In any of the plots, right-click on a point to exclude the point from the line fitting. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 201 This may be evident from direct comparison of the kinetic fits, but will not be immediately apparent in the thermodynamic analysis. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 202 10 Thermodynamic analysis 10.2 Performing thermodynamic analysis Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 203: Conventions And Background
B determined over the same sensor surface, in order to calculate the free B concentrations in the samples. 11.1.2 Evaluation principles The equilibrium constant for a 1:1 interaction is given by ⋅ free free ---------------------------- - – – ------------------------------------------------------- - Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 204: Requirements For Affinity In Solution
Refer to Chapter 5 for details of how to construct methods in Method Builder. Determination of affinity in solution is not supported by a wizard. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 205: Evaluation Of Affinity In Solution
Samples that lie outside the range of the calibration curve are not shown. Right click on calibration points to exclude the points from the curve. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 206 ConcB. This value should be the same as or close to the value entered for the variable ConcB in the method. The calculated K value is shown in the panel below the table. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 207 Appendices Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 208 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 209: A.1 Exporting Data
.xls) containing separate worksheets for the file properties and report point table. The audit trail is also exported to a separate worksheet if it is present when the GxP module is installed (see the separate Biacore T100 GxP Handbook). Export from the Evaluation software an Excel spreadsheet file (extension .xls) containing separate worksheets for the file properties and for tabulated data for all evaluation items where appropriate (i.e.
Page 210: A.2 Importing Data
Import programs and data files for use with import are the responsibility of the user. For each import function, a check box allows the import program to be started automatically without user intervention. If the respective box is not checked, the Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 211 Figure A-1. Part of an XML import file from a method for heterogeneous analyte kinetics displayed in an XML-compatible editor. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 212 Position specification and must be placed in the microplate or reagent rack before the run can be started. Details of the required import file format can be investigated further by examining a file created with the Menu:Export Positions command. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 213: A.2.2 Evaluation Software
Evaluation Software The Evaluation Software supports import of model definitions for kinetics and affinity evaluation. Model files for import should be obtained from GE Healthcare or created by exporting models from another installation. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 214 Appendix A Data import and export A.2 Importing data Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 215: B.1 Affinity In Solution
Predefined evaluation variables are included for the evaluation purpose Affinity in solution: The Variable Settings is different for the two assay steps, so that only the relevant variables are entered at run-time for each assay step. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 216: B.2 Calibration-Free Concentration Analysis
µl) may differ significantly at the lowest flow rate. Sample solution and flow rate are set as method variables. For evaluation variables, the evaluation purpose is set to Calibration-free conc and predefined variables MW, D(20°C), Blank and Dilution are required. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 217: B.2.3 Variable Settings
Method examples and recommendations Appendix B B.2.3 Variable settings Sample cycles All the variables for assay step Sample are set at run time, except for the method variable Blank which is set to No in the method. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 218: B.2.4 Setup Run
(MW), diffusion coefficient at 20°C (D(20°C) and dilution factor (Dilution). Only flow rates and dilution factor are required for assay step Blank: these values must be the same as the flow rates used for samples. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 219: B.3 Gst Kinetics
General Settings. Detection settings Multi and Single are not recommended. The method involves 5 conditioning cycles with injection of recombinant GST (provided in the GST Capture Kit from GE Healthcare) over both flow cells followed by regeneration: The sample analysis cycle uses two capture injections, one to capture...
Page 220 Note: If you change the detection setting to Multi, you will need to include additional capture injections for the active surfaces in flow cells 3 and 4. Do not use detection setting Single without removing the first capture injection. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 221: B.4 Inject And Recover
The cycle for recovery of bound analyte contains just one single InjectAndRecover command. Additional commands are usually not needed, since the recovery component of the InjectAndRecover command serves as a regeneration step (see Section 5.6.1). Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 222 B.4 Inject and Recover All parameters in the InjectAndRecover command are fixed: check the appropriate boxes in the Method Variables list if you want to use variable parameters. There are no evaluation variables for this command. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 223: B.5 Kinetics Heterogeneous Analyte
The recommended method for liposome capture on Sensor Chip L1 uses one conditioning cycle consisting of two injections of 40 mM octylglucoside, followed by three start-up cycles using a dummy sample before the actual samples are run. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 224: B.7 Lmw Kinetics And Lmw Screen
This assay step is connected to a cycle type that includes 8 Solvent Correction commands for injection of 8 different solvent concentrations (see Section 6.7). Note: In Biacore T100 Control Software version 2.0 and later, solvent correction is included in the Kinetics/Affinity and Thermodynamics wizards.
Page 225 If you require a regeneration step, it is advisable to include regeneration after both the sample and the carry-over injections. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 226: B.8 Nta Kinetics
Ni and one for the poly-His tagged ligand. The Biacore method provided is designed for kinetic or affinity determinations, but may be readily modified for other applications that use Sensor Chip NTA. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 227: B.9 Single-Cycle Kinetics
Single cycle kinetics with 5 sample concentrations per cycle. Note that with this command type, the evaluation purpose is fixed as Kinetics/Affinity and the number of predefined variables for sample concentration corresponds automatically to the number of sample concentrations per cycle. Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 228 Appendix B Method examples and recommendations B.9 Single-cycle kinetics Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 229: Index
142 connecting to cycle type 84 calculated concentrations 141 recurrence 84 calculated parameters 192 replicates 84 calibration curve 62 run order 84 calibration curves 205 auto scale 20 calibration-free concentration automatic positioning 41 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 230 149 results 74 DMSO 67 control samples 36 drift 191 binding analysis 60 dual detection 80 concentration analysis 64 dual inject 89 kinetics/affinity 69 dummy sample 35 plot 110 control software Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 231 Excel 209 histidine tagged ligands 226 export to XML 209 exporting data 209 exporting plots 108 if...then command 87 exporting rack positions 42 immobilization 46 exporting the report point table 210 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 232 68 ligand immobilization 46 multi detection 80 limit guides 172 multi-component complex 59 linear fit 129 multi-cycle kinetics 66 linked reactions 72 multiple data sets 156 control experiment 73 multiple ligand densities 162 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 233 18 export 210 printing rack positions 42 report point window 112 printing results 18 report points 11 printing wizard templates 42 adjustment for molecular weight 125 editing in control software 19 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 234 225 standard deviation 136 SD 136 standard error 151 selecting data for thermodynamic standby 21 evaluation 199 start-up cycles 35 selector bar 17 in regeneration scouting 52 selector button 122 statistical parameters 168 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 235 186 uniqueness 165 unknown samples 139 unzoom 20 user-defined caption 107 user-defined variables 92 U-value 169 van’t Hoff equation 197 variable settings 96 variables 78 entering values at run time 98 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 236 Biacore T100 Software Handbook BR-1006-48 Edition AE...
Page 238 Björkgatan 30 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these 751 84 Uppsala terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.

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