Cayman Chemical | Analysis | FPIA

FPIA 384 Wells

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This is the Cayman Chemical Company 384 Well FPIA page. It contains information, Excel workbooks, and other tools useful for anyone performing FPIA 384 well assays.

If you have questions on the use of our tools, or feel something is missing, please contact Technical Services.

The Workbooks

Currently, Cayman Chemical uses two Excel workbooks to perform 384 well FPIA analysis. Both are available for your download and use.

Download

Your downloading these files implies acceptance of the license agreement listed below and present in each workbook.

FPIA384Triple ~ 1 Mb XLS formatted for assays performed in triplicate
FPIA384Double ~ 1 Mb XLS formatted for assays performed in duplicate

Trouble downloading? Right-click the link and select "Save Target As..."

These instructions are repeated on the first sheet of each workbook.

FPIA 384 Well Triple Worksheet

FPIA 384 Well Double Worksheet

Definition of Key Terms

Term	Definition
mP(max)	Maximum fluorescence polarization for the unbound tracer.
Std0# (Standards)	Standards #1 through #8 made by serially diluting Std01 seven times.
Smpl# (1:*)	Samples and their associated dilution factors

Workbook Sheets

Analysis

LAYOUT

Contains sample names and associated dilution factors.

ASSAY

Pull-down menu for selection of the FPIA being performed.

G-Factor

The value of the G-factor for the assay being performed.

BKG-PARL

Fluorescence of the sample, prior to addition of the fluorescent reagent, with the emission polarizer set parallel to the excitation polarizer.

BKG-PERP

Fluorescence of the sample, prior to addition of the fluorescent reagent, with the emission polarizer set perpendicular to the excitation polarizer.

PARALLEL

Fluorescence of the sample, after addition of the fluorescent reagent, with the emission polarizer set parallel to the excitation polarizer.

PERPENDICULAR

Fluorescence of the sample, after addition of the fluorescent reagent, with the emission polarizer set perpendicular to the excitation polarizer.

Calculated using the following equation:

mP = 1000 x	(I_parl - I_perp)
	(I_parl + I_perp)

LOGIT

The logit for each cell is computed using the formula: ln[(mP/mP_max)/((1 - (mP/mP_max))]. This technique 'linearizes' the values to facilitate regression analysis.

CONC

The concentration for each cell is calculated based on the LOGIT value, constants from the log-regression curve (see StdCurve section), and the dilution factor from the LAYOUT section.

STANDARDS

The 'Avg', 'mP/mP_max', 'Logit', and 'Recalc' values are calculated for each concentration value. Only the value of 'Std01' must be entered; subsequent values are calculated automaticallly. 'Recalc' are the standard concentration values calculated from the regression constraints.

Samples

This table is compiled from the preceding sections. It includes

Conc: average of the replicate concentrations from the CONC section. Data outside the 20-80% mP/mP(max) range are highlighted.
mP/mP_max: average of the replicate values from the mP/mP_max section expressed as a percentage.
Dilution: the dilution factor, for the sample, from the LAYOUT section.
StdDev: the standard deviation of the replicate concentration values.
%CV: the coefficient of variance of the replicate concentration values.
SEM: the standard error of the mean for the replicate concentration values.

STATS

'y-int' and 'slope' are obtained from the log-regression fit of the calibration data.

CURVE

The IC20, IC50, and IC80 values are calculated. The IC20 & IC80 are the upper and lower detection limits of the standard curve, respectively. The IC50 is a quality control paramater (given as 50% mP/mP_max).

FILENAME

The filename given to the Excel workbook.

VERSION

The version of the Excel workbook.

StdCurve

This is the calibration curve of the concentration standards plotted versus the logit(mP/mP_max) values. The data are fit to a logarithmic regression line with the general equation: y = mln(x) + b.

Instructions

The worksheets were designed to be as “hands-off” as possible. The following instructions assume you have loaded your plate according to one of the plate layouts shown on the Workbooks sheet, and have the data in a form that can be imported (or pasted) directly into a spreadsheet. To analyze your data:

Select the assay you are running from the pull down menu.
Input your sample names into the appropriate cells.
Input the sample dilutions into the appropriate cells in the format shown (e.g., 1:5).
Paste/type your data into the RAW DATA section (starting in cell B22) of the Analysis worksheet.

Select the “Values” option in the “Paste Special…” dialog box to maintain formatting. All concentration values, and relevant statistics, are automatically calculated. The Samples table is then filled in with the data. All averages and statistics are calculated based on all replicates for each sample. It is the responsibility of the individual researcher to decide if any data can be eliminated. Caution should be exercised, however, when doing this! Cayman Chemical has examined statistical methods to test for the presence of outliers in EIA assays. We find that individual replicates are rarely flagged as outliers, and cannot be eliminated from the experiment.

NOTE: The sole purpose of the data contained in this workbook is to demonstrate the functions of the individual spreadsheets.

Copyright and Disclaimer

Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:

Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer.
Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution.

THIS SOFTWARE IS PROVIDED BY CAYMAN CHEMICAL COMPANY, INC. “AS IS” AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL CAYMAN CHEMICAL BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.

The views and conclusions contained in the software and documentation are those of the authors and should not be interpreted as representing official policies, either expressed or implied, of Cayman Chemical Company, Inc.