Tutorial 0: Introduction to Scrubber

Scrubber2 Version 2.0a January 2006

This tutorial introduces Scrubbers layout, features, and how it should be used to process and analyze rapidly the biosensor data collected on Biacore platforms. Its simple, user-friendly interface guides you through a series of data transformations that clean up or scrub data in seconds. Scrubber is also a data analysis tool that can be used to fit equilibrium binding responses to extract affinities and percent bound values or to fit kinetic data to calculate the rate constants and affinities. Scrubber can also provide an estimate of the dissociation rate constant by fitting a single exponential decay to dissociation phase data. Scrubbers versatile graphics allow the user to view data in various ways at any stage of processing and analysis, customize graphs with respect to color, font, and size, and copy and paste them into other documents such as MS Word and Powerpoint to facilitate report writing and slide presentations.

Each tutorial contains a detailed description of the data processing and analysis transformations using red arrows and numbered yellow circles to guide you through each step. Commands and options in bold are found in the software.

Opening Scrubber

The Data page is the default opening page and is one of twelve tab pages that are intended to be visited sequentially from left to right. The function of each page is described in this tutorial.

plot window
loaded data are displayed here

Click the Load button on the toolbar to load files in one of three formats: 1) Biosensor (*.blr) files are either Biacore result files or BIAevaluation files. 2) Data (*.txt) files are text files exported from BIAevaluation. 3) Scrubber (*.scb) files are files that were previously processed in Scrubber.

Click the down arrow to reveal the range of available File types. View files by one of the options shown, select a file by name, and click the Open button.

Opening a Data File

A grid appears on the left, in which each cell represents an individual interaction analysis cycle. Cells are intended to be read down in vertical columns starting at A1. The number of available cells equals the number of injection cycles in the data set.

An overlay plot depicts raw data with default axis labels of Time (X) and Response (Y).

By default, the grid is a ten-row array (numbered 1, 2, 3, 10) with up to six columns in view (labeled alphabetically). The grid is intended to mimic a thermo_A autosampler rack. If the data set comprises more than 60 analyte injection cycles, you can use a scroller to move to additional columns at the right (1). Note that the scroller only appears when data sets of more than 60 analyte injections are loaded). Also, you can customize the number of rows in the grid by toggling the up/down arrow key (2), e.g., 12 rows mimics the 12 x 8 array of a 96-well micro-titer plate.

Loading a Previously Saved Scrubber File

The full complement of data processing steps are retained, as indicated by the tab page labels becoming bolded black for all activated pages blue for the page currently displayed. The tab label in the current page changes from blue to bolded blue when the transformation has been applied. By default, the Compound and Result tabs retain their unbolded labels. Each cell in the grid represents an individual analyte injection.

In this example, the concentration of each analyte injection (cell) in the grid has been specified using a code. Use the Stock conc and Dilution factor edit boxes to define a concentration series that correlates with a numerical code entered in the grid. A two-fold serial dilution of a 400 micromolar stock solution is defined above, where the numbers in the grid (2, 3, 4, 5, 6, etc.) code for successive dilutions of the stock solution (1), e.g., 1 = 400 M (Stock conc), 2 = 200 M, 3 = 100 M, 4 = 50 M, 5 = 25 M etc. Other codes used in the grid are 0 = buffer (blank) and d = DMSO sample (to denote a calibration series that is used when working in buffers containing high refractive index solvents, such as DMSO). Alternatively, enter an analyte concentration into the relevant cell based on the cycle number (e.g., cell B3 = cycle 13) using m, u, n, and p for units of milli-, micro-, nano-, and picomolar respectively,e.g., 10m = ten millimolar, 10u = ten micromolar, 10n = ten nanomolar, 10p = ten picomolar. Since analyte concentrations entered directly into the grid are independent of the Stock conc and Dilution factor edit boxes that define the indirect code-wise format, the grid can support mixtures of both formats for different analytes.

Right-Clicking on a Plot Window

A menu with various display options is revealed.

Legend

Activate Zoom functions by first drawing a box around an area of interest using the left mouse button (click+drag).

Linear scale
uncheck the scale option Log

Labels

Log scale
default

Activate Hide functions by first drawing a box around the data points that you want to discard using the left mouse button (click+drag). This tool is a useful way of removing air spikes.

Activate the Properties dialog to customize the way in which graphs are displayed.

Copy data allows copying of the responses in a numerical form so the data can be opened and manipulated in a spreadsheet software such as Microsoft Excel.

Graph properties

The scale of the plots can be set automatically or manipulated by the user to show the responses over different surfaces at the same scale.

The axes, lines and points can be labeled and a descriptive title can be used for the plot. An inset of the kinetic parameters or text can be positioned within the plot either at the left or the top. The values can be enclosed in a box.

The font size, color and shape can be manipulated.

The graphic representation of the plots can be manipulated by selecting the line thickness for the axis, the data curves and the model. The error for repeat measurements in binding isotherms can also be included. Graphs can be shown as curves or points. The scale of the points can also be adjusted. The zero lines can also be shown in the plot for visual reference. 6

The graphs can be copied as bitmap files into software such as MS Word and Powerpoint. The user can define the quality of the copied file using the Copy Width and Copy Height functions. The default values are 1000X600 dpi, but these settings can be changed.

Data Transformations: Y-Zero, Crop, X-Align, Reference, and Blank

A pair of before (left) and after (right) graphs appear on each of these five pages. The result of the preceding active page provides the source data (left panel) for the current transformation (right panel), which in turn provides the input data for the next active page. Drag the pair of vertical time lines (green = start and red = stop) to select appropriate limits for each data transformation. Their new positions update in their respective edit boxes. This page Y-averages data to zero to establish a baseline, so choose time limits prior to the injection start. Crop the data to focus on the interaction. Panels depicting the pair of before and after graphs are named in blue according to the most recently applied data transformation (from left to right along the tab pages). In this example, the result of the Zero page (the preceding page) provides the source data for the current Crop page. The number of data points in a data set can be decreased to speed up the data processing. Define the number of seconds to average data points over that time frame. Reducing the number of data points does not affect the outcome of the kinetic analysis. Align all curves to zero on the X-axis to denote a common injection start time of zero seconds. With appropriate time limits, most if not all curves should align to zero at the base of their steepest slopes by simply checking the Align box. However, some curves with ambiguous start times may misalign. Manually align individual curves by toggling the left/right arrow key in the Set injection time box.

apply

reduce the number of points

align individual curves

Reference the data by selecting a suitable reference flow cell using the radio buttons. The right panel depicts reference-subtracted responses for all other flow cells.

Double reference the data within each Fc by subtracting a buffer (blank) response. In this example, an Average curve has been computed from all blank curves in the left panel and subtracted from all Referenced curves (corresponding to the right panel of the preceding page), giving the Blanked responses in the right panel of this page. For blank injections that change during an experiment, the Closest blank injection can be subtracted from each Referenced curve. 7

Solvent Correction
For running buffers that contain a high-refractive index solvent such as DMSO, the Dmso page allows you to correct for mismatched refractive index increments between running and sample buffers. A calibration plot is constructed in the upper window using data derived from injections marked d (DMSO samples) on the Data page.

Fit a linear trend line. A polynomial may be more appropriate in describing a solvent concentration series spanning a wider range.

Unreferenced responses obtained across the reference flow cell (X-axis) are plotted against referenced responses from all other flow cells (Y-axis) and fit to the chosen trend line. Flow cells are discriminated by color.

This plot is constructed from samples marked d on the Data page.

Select a portion of the association phase using the green and red vertical times lines. Specify the time at which the injection ends using the blue vertical line. Re-positioning these lines updates the times recorded in their respective edit boxes (1, 2, and 3).

Black triangles indicate where the analyte samples lie within the solvent range explored. The correction cannot be applied properly to samples lying outside this range. If the calibration is too narrow, repeat the analysis using a solvent series that spans a wider concentration range.

Compound Page
Name the reactants on this page and specify which injection belongs to which analyte when multiple analytes are being analyzed. The default mode assumes that only a single analyte is being studied. Customize this page as demonstrated below. Unscaled Toggle between black (default) and color by Fc modes. The color scheme mimics that of a Biacore read-out. Scaled

This option scales RU by an analytes MW (as entered in the Analyte table)

Toggle up/down arrow key to add/delete analytes

There are three color options for Analytes: 1) All black (default mode) 2) Color by analyte 3) Color by analyte concentration (by default, DMSO curves are highlighted in blue)

This grid mimics the concentration grid on the Data page, but here it is used to specify which interaction cycle belongs to which analyte when multiple analytes are being studied. By default, all cells are assigned to Analyte 1. Customize the grid by assigning an analyte number (1, 2, 3, 4, ) to cells belonging to individual analytes.

Equilibrium Analysis
Select equilibrium positions on the Bound page by averaging values within user-specified limits. Click on Calc to construct a plot of Response vs. Concentration in the right panel. By default, the X-axis (representing analyte concentration in Molar units) is expressed as a log scale and a legend appears, naming the analyte.

On the Affinity page, derive KD and % Bound values (for protein-based assays) from the equilibrium binding responses defined on the previous page by applying one of two reaction models, either a single-site model (A+B=AB) or a two-independentsites model (A+B=AB, A+C=AC, where A is the injected analyte and B and C are different ligand-binding sites). Fit Rmax globally or optimize it locally for each binding site and/or analyte by clicking L=local and F=float. Since % Bound values are based only on the high-affinity site, they are given in parentheses for a two-sites model. The responses can be normalized to the calculated Rmax to display the binding isotherm in terms of percent capacity.

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Kinetic Analysis
In the Kinetics window, data sets can be fit to derive the kinetic parameters of an interaction. The injection start time is marker by the green line while the end time is marker by the blue line. Alternatively the start and end times can also be entered into the corresponding boxes. Data sets can be fit using one of three sets of conditions 1) the kd only, where the dissociation phase is fit to a single exponential decay to calculate the dissociation rate and the half life of the complex (t1/2 = ln2/kd), 2) the ka and the kd of an interaction based on a simple bimolecular model (A+B=AB), or 3) the km, ka and kd for mass-transport limited reactions, where Ao=A+B=AB.

Data can be fit to a selected model by clicking the appropriate radio button. Initial estimates of the kinetic parameters appear a starting point for the fitting process. Selecting the Fit option fits the selected model to the data.

At the end of the fit, parameters such as the km, ka, kd, Rmax, KD, t and the residuals of the fit are tabulated, depending on the model used. These can be viewed by scrolling across the screen as indicated.

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During the fitting process, parameters can be fixed or floated accordingly. To fix a kinetic variable, the edit box is highlighted and the Fix icon is pressed. The font color of a fixed parameter becomes red. Selecting the Float icon floats the value of the parameter. The font color of a floated value changes from red to black.

Selecting the Options icon, opens the Kinetic options menu. Some of the options incorporated in this window include adjustment of the beginning and end times of the injections, fitting of the bulk refractive index changes in the responses, linking kinetic parameters for different reactions and normalizing for different Rmax values. These parameters can be fixed or floated using the buttons on the top of the screen.

The injection start times can be adjusted using the Begin window. The start times can be fit together for all injections by selecting the Fit option (left panel). Alternatively each injection can be fit to its own start time using the Separate option (right panel). Under this option the column called Begin Inj can be selected and the start times of each injections can either be fit or floated. Values for the fixed times change into red and values for the floated times are black.

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The injection end times can be adjusted using the End window. Similarly to the start times of the injections, the end times can be fit together for all injections (applying the Fit option on the left panel) or they can be fit separately using the Separate option (right panel). The Begin Inj column can be selected for the start times of each injection to be fixed or floated using the appropriate icons on the top of the window. The fixed values change into a color-font and floated values remain black.

Applying the Bulk RI function corrects for bulk refractive index changes in the binding traces. The Inj Baseline column can be selected and refractive index changes for all the injections can either be fixed or floated. The corrected responses appear under in the Results page.

Injections which share the same parameters can be connected together using the Link function to globally fit them. Assignment of an injection number under a kinetic parameter column links the kinetics together for different injections. For example, the km of injection 43 is linked to the km of injection 47, by assigning the number 43 under the km column across from injection 47. As a result the km value for injection 47 will be the same as the value calculated for injection 43.

Under the Options panel, the Rate upper limit, the End chisq change, the Max number interactions and the Update display (cycles) can be defined. Additional options to Restart if stalled, Display as %Rmax and Show zero conc curves are also included.

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Result Page
This page offers numerous display options that are activated by selecting various permutations of buttons and pull-down menus in the toolbar. Alternatively, you can customize Graph Properties via a pop-up menu that is activated when you right-click on a plot. Switch all buttons ON to view plots of Response vs. Concentration and Response vs. Time split by Fc and analyte. The red traces represent the fit of the kinetic model to the data.

Data can be viewed in singleor multi-channel modes.

Switch Lgnd button OFF to construct overlay plot of all Fcs.

Alternate display

Identify ligands by color on the Compound page.

This overlay plot is in Label mode. Right-click to return to Legend mode.

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Hide Options
Selecting the Hide option allows the exclusion of data from an analysis.

Uncheck the analyte you want to exclude from the analysis and select the Done button

Hidden responses are marked in red

The hidden analytes are greyed out

Uncheck the flowcell you want to exclude from the analysis

Uncheck the Injection you want to exclude from the analysis

Uncheck the concentration you want to exclude from the analysis

Uncheck the curve you want to exclude from the analysis

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Advanced Tools
Data destined for kinetic analysis should be analyzed at a high data collection rate to generate the maximum number of available data points. In the default mode, only a fraction of these are displayed to speed up the data transformations. You can switch from the standard mode to an All point mode for improved resolution, which is particularly useful when making fine adjustments on the Align page or when identifying a sharp spike that is perturbing the scale of a graph. Another advanced tool is the Despike option, which allows you to remove spikes from data instantly. Use it with caution because despiking changes the short-term noise structure of the data, which can affect the analysis when the signal-to-noise ratio is low. Both of these tools are found in the toolbar.

STANDARD resolution

MAXIMUM resolution

Spikes removed instantly 16

Save Options
Save an entire method as a Scrubber (*.scb) file at any stage of the analysis. Save graphs displayed on the Result page as *.txt files. This option is inactive on other pages.

Scrubber files provide useful templates for instantly scrubbing new data sets. Simply load a new data set onto a pre-loaded Scrubber file and turn the Clear method option OFF to apply the full complement of data transformations to the new data.

When saving a Scrubber file, check the Save associated data option so that the program knows where to find it. Save Response vs. Time plots as either: 1) Biosensor data files, which are compatible with spreadsheets, such as Excel and BIAevaluation.

Open Biosensor data files in BIAevaluation.

Save Response vs. Concentration plots as Binding data files (*.txt) and open them in Excel.

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Overlay plot of same data in BIAevaluation.

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Print Options

Enter details about the experiment here.

A typical print-out

METHOD DETAILS

FIT RESULTS RESULT PAGE

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