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This tutorial introduces Scrubbers layout, features, and how it should be used toprocess and analyze rapidly the biosensor data collected on Biacore platforms. Itssimple, user-friendly interface guides you through a series of data transformationsthat clean up or scrub data in seconds. Scrubberis also a data analysis tool thatcan be used to fit equilibrium binding responses to extract affinities and percentbound values or to fit kinetic data to calculate the rate constants and affinities.Scrubbercan also provide an estimate of the dissociation rate constant by fitting asingle exponential decay to dissociation phase data. Scrubbersversatile graphicsallow the user to view data in various ways at any stage of processing andanalysis, customize graphs with respect to color, font, and size, and copy and

paste them into other documents such as MS Wordand Powerpoint to facilitatereport writing and slide presentations.

Scrubber2Version 2.0aJanuary 2006

Tutorial 0: Introduction to Scrubber

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

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plot windowloaded data are displayed here

The Data page is the default openingpage and is one of twelve tab pagesthat are intended to be visitedsequentially from left to right. The

function of each page is described inthis tutorial.

Click the Load button on the toolbar to load files in one of three formats:

1) Biosensor (*.blr) files are either Biacoreresult files or BIAevaluationfiles.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 therange of available File types. Viewfiles by one of the options shown,select a file by name, and click the

Open button.

Opening Scrubber

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Opening a Data File

A grid appears on the left, in which

each cell represents an individual

interaction analysis cycle. Cells areintended to be read down in vertical

columns starting at A1. The numberof available cells equals the number

of injection cycles in the data set.

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 additionalcolumns 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 bytoggling the up/down arrow key (2), e.g., 12 rows mimics the 12 x 8 array of a 96-well micro-titer

plate.

An overlay plot depicts raw data with default

axis labels of Time (X) and Response (Y).

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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 tablabel in the current page changes from blue to bolded blue when the transformation has beenapplied. By default, the Compound and Result tabs retain their unbolded labels.

Loading a Previously Saved Scrubber File

Each cell in the grid represents an

individual analyte injection.

In this example, the concentration of each analyte injection (cell) in the grid has beenspecified using a code. Use the Stock conc and Dilution factor edit boxes to define aconcentration series that correlates with a numerical code entered in the grid. A two-foldserial dilution of a 400 micromolar stock solution is defined above, where the numbers in thegrid (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 thegrid are 0 = buffer (blank) and d = DMSO sample (to denote a calibration series that is usedwhen 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 pico-molar respectively,e.g., 10m = ten millimolar, 10u = ten micromolar, 10n = ten nanomolar,

10p = ten picomolar.

Since analyte concentrations entered directly into the gridare independent of the Stock concand Dilution factor edit boxes that define the indirect code-wise format, the grid can supportmixtures of both formats for different analytes.

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A menu with various display options is revealed.

Activate the

Properties dialogto customize the

way in which

graphs aredisplayed.

Right-Clicking on a Plot Window

Activate Zoomfunctions by firstdrawing a box

around an area ofinterest using the

left mouse button(click+drag).

Copy data allows copying of theresponses in a numerical form so the

data can be opened and manipulatedin a spreadsheet software such as

Microsoft Excel.

Activate Hide functions by firstdrawing a box around the datapoints that you want to discard

using the left mouse button(click+drag). This tool is a useful

way of removing air spikes.

Legend

Labels

Linear scaleuncheck the Logscale option

Log scaledefault

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Graph properties

The scale of the plots can beset automatically or

manipulated by the user toshow the responses over

different surfaces at thesame scale. The axes, lines and points

can be labeled and a

descriptive title can be usedfor the plot.

The graphic representation of

the plots can be manipulatedby selecting the line thicknessfor the axis, the data curves

and the model. The error forrepeat measurements in

binding isotherms can also beincluded. Graphs can be

shown as curves or points.The scale of the points canalso be adjusted. The zero

lines can also be shown in theplot for visual reference.

The graphs can be copied as bitmapfiles into software such as MS Wordand Powerpoint. The user can definethe quality of the copied file using the

Copy Width and Copy Heightfunctions. The default values are1000X600 dpi, but these settings can

be changed.

An inset of the kineticparameters or text can be

positioned within the ploteither at the left or the top.The values can be enclosed

in a box.

The font size, color andshape can be manipulated.

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

A pair of before (left) and after (right) graphs appear oneach of these five pages. The result of the preceding activepage 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 verticaltime lines (green = start and red = stop) to selectappropriate limits for each data transformation. Their newpositions update in their respective edit boxes. This page

Y-averages data to zero to establish a baseline, so choosetime 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 blueaccording to the most recently applied data transformation

(from left to right along the tab pages). In this example, theresult 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 dataprocessing. Define the number of seconds to average data

points over that time frame. Reducing the number of datapoints does not affect the outcome of the kinetic analysis.

Align all curves to zero on the X-axis to denote a commoninjection start time of zero seconds. With appropriate timelimits, 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 theleft/right arrow key in the Set injection time box.

Reference the data by selecting a suitable reference flowcell 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 abuffer (blank) response. In this example, an Average curvehas been computed from all blank curves in the left paneland subtracted from all Referenced curves (corresponding

to the right panel of the preceding page), giving theBlanked responses in the right panel of this page. Forblank injections that change during an experiment, the

Closest blank injection can be subtracted from eachReferenced curve.

apply

alignindividualcurves

reduce thenumber ofpoints

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For running buffers that contain a high-refractive index solvent such as DMSO, the Dmso pageallows you to correct for mismatched refractive index increments between running and samplebuffers. A calibration plot is constructed in the upper window using data derived from injections

marked d (DMSO samples) on the Data page.

Solvent Correction

Unreferenced responses obtained acrossthe reference flow cell (X-axis) are plotted

against referenced responses from all otherflow cells (Y-axis) and fit to the chosen trend

line. Flow cells are discriminated by color.

Fit a linear trend line. A polynomial

may be more appropriate indescribing a solvent concentration

series spanning a wider range.

Black triangles indicate where the analytesamples lie within the solvent range

explored. The correction cannot be appliedproperly to samples lying outside thisrange. If the calibration is too narrow,

repeat the analysis using a solvent seriesthat spans a wider concentration range.

Select a portion of the association phaseusing the green and red vertical times lines.

Specify the time at which the injection endsusing the blue vertical line. Re-positioningthese lines updates the times recorded in

their respective edit boxes (1, 2, and 3).

1 2 3

1 2 3This plot is constructed

from samples marked don the Data page.

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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.

Compound Page

Unscaled Scaled

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

Toggle between black (default)and color by Fcmodes. The colorscheme mimics that of a Biacoreread-out.

This grid mimics the concentration grid on theData page, but here it is used to specify which

interaction cycle belongs to which analyte whenmultiple analytes are being studied. By default,

all cells are assigned to Analyte 1. Customizethe grid by assigning an analyte number (1, 2, 3,

4, ) to cells belonging to individual analytes.

Toggle up/down arrow keyto add/delete analytes

1 2 3

There are three color options for Analytes:

1) All black (default mode)

2) Color by analyte3) Color by analyte concentration (by default,

DMSO curves are highlighted in blue)

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Equilibrium Analysis

On the Affinity page, derive KD and% Bound values (for protein-basedassays) from the equilibrium bindingresponses defined on the previous

page by applying one of two reactionmodels, either a single-site model

(A+B=AB) or a two-independent-sites model (A+B=AB, A+C=AC,where A is the injected analyte and

B and C are different ligand-bindingsites). Fit Rmax globally or optimize it

locally for each binding site and/or

analyte by clicking L=local andF=float. Since % Bound values arebased only on the high-affinity site,they are given in parentheses for a

two-sites model. The responses canbe normalized to the calculated

Rmax to display the bindingisotherm in terms of percent

capacity.

Select equilibrium positions

on the Bound page by averagingvalues within user-specified limits.

Click on Calc to construct a plot of

Response vs. Concentration in theright panel. By default, the X-axis

(representing analyte concentrationin Molar units) is expressed as a log

scale and a legend appears, namingthe analyte.

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Kinetic Analysis

1 2 3

In the Kinetics window, data setscan be fit to derive the kineticparameters of an interaction. The

injection start time is marker by the

green line while the end time ismarker by the blue line. Alternativelythe start and end times can also beentered into the corresponding

boxes. Data sets can be fit using oneof three sets of conditions 1) the kdonly, where the dissociation phase isfit to a single exponential decay tocalculate 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 simplebimolecular model (A+B=AB), or 3)

the km, ka and kd for mass-transportlimited reactions, whereAo=A+B=AB.

Data can be fit to a selected modelby clicking the appropriate radio

button. Initial estimates of the kineticparameters appear a starting point

for the fitting process. Selecting the

Fit option fits the selected model tothe data.

At the end of the fit, parameters such

as the km, ka, kd, Rmax, KD, t and theresiduals of the fit are tabulated,depending on the model used. These

can be viewed by scrolling acrossthe screen as indicated.

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During the fitting process,

parameters can be fixed or floatedaccordingly. To fix a kinetic variable,

the edit box is highlighted and the

Fix icon is pressed. The font color ofa fixed parameter becomes red.

Selecting the Float icon floats thevalue of the parameter. The font

color of a floated value changes fromred to black.

Selecting the Options icon, opensthe Kinetic options menu. Some ofthe options incorporated in this

window include adjustment of thebeginning and end times of the

injections, fitting of the bulk refractiveindex changes in the responses,

linking kinetic parameters fordifferent reactions and normalizingfor different Rmax values. These

parameters can be fixed or floatedusing the buttons on the top of the

screen.

The injection start times can beadjusted using the Begin window.The start times can be fit together forall injections by selecting the Fitoption (left panel). Alternatively eachinjection can be fit to its own starttime using the Separate option (rightpanel). Under this option the columncalled Begin Inj can be selectedand the start times of each injectionscan either be fit or floated. Values for

the fixed times change into red andvalues for the floated times areblack.

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The injection end times can beadjusted using the End window.Similarly to the start times of the

injections, the end times can be fittogether for all injections (applying

the Fit option on the left panel) orthey can be fit separately using the

Separate option (right panel). The

Begin Inj column can be selected forthe start times of each injection to befixed or floated using the appropriateicons on the top of the window. The

fixed values change into a color-fontand floated values remain black.

Applying the Bulk RI functioncorrects for bulk refractive index

changes in the binding traces. The

Inj Baseline column can beselected and refractive index

changes for all the injections caneither be fixed or floated. The

corrected responses appear under inthe Results page.

Injections which share the sameparameters can be connected

together using the Link function toglobally fit them. Assignment of aninjection number under a kinetic

parameter column links the kinetics

together for different injections. Forexample, the km of injection 43 islinked to the km of injection 47, by

assigning the number 43 under thekm column across from injection 47.As a result the km value for injection

47 will be the same as the valuecalculated for injection 43.

Under the Options panel, the Rateupper limit, the End chisq change,

the Max number interactions andthe Update display (cycles) can bedefined. Additional options to Restartif stalled, Display as %Rmax andShow zero conc curves are alsoincluded.

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This page offers numerous display options that are activated by selecting various permutations ofbuttons and pull-down menus in the toolbar. Alternatively, you can customize Graph Properties viaa pop-up menu that is activated when you right-click on a plot.

This overlay plotis in Labelmode. Right-clickto return toLegend mode.

Switch Lgnd buttonOFF to constructoverlay plot of all Fcs.

Identify ligands by coloron the Compound page.

Alternatedisplay

Data can be viewed in single-or multi-channel modes.

Switch all buttons ON to view

plots of Response vs.Concentration and Responsevs. Time split by Fc andanalyte. The red tracesrepresent the fit of the kinetic

model to the data.

Result Page

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Hide Options

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

Uncheck the

flowcell youwant to excludefrom the analysis

Uncheck the

Injection youwant to excludefrom the analysis

Uncheck the

curve you want toexclude from theanalysis

Uncheck the

concentrationyou want to

exclude from theanalysis

Uncheck the analyte you want to excludefrom the analysis and select the Donebutton

Hidden responsesare marked in red

The hidden

analytesare greyedout

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Advanced Tools

Data destined for kinetic analysis should be analyzed at a high data collection rate to generate themaximum 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 Allpoint 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. Anotheradvanced tool is the Despike option, which allows you to remove spikes from data instantly. Use itwith 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

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Save Options

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0

5

10

15

20

25

-200 -150 -100 -50 0 50 100 150 200 250 300 350

Scrubber files provide usefultemplates for instantly scrubbingnew data sets. Simply load a new

data set onto a pre-loaded

Scrubber file and turn the Clearmethod option OFF to apply thefull complement of data

transformations to the new data.

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

Open Biosensor data files in BIAevaluation.

Save an entire method as a

Scrubber (*.scb) file at anystage of the analysis.

Save graphs displayed on the

Result page as *.txt files.This option is inactive on

other pages.

When saving a Scrubber file,check the Save associateddata option so that theprogram knows where to findit.

Overlay plot of

same data inBIAevaluation.

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Save Response vs. Timeplots as either:

1) Biosensor data files, which arecompatible with spreadsheets, such asExceland BIAevaluation.

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Print Options

Enter details about

the experiment here.

A typical print-out

RESULT PAGE

METHOD DETAILS

FIT RESULTS

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