ProStain™
Protein Quantification Kit(version D2)
Catalog No. 15001

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of
any kind, either explicit or implied. Information may be changed or updated in this manual at anytime.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and internationaltreaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2008 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
The purification column used in this kit is covered under US patent 005618418A.
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TABLE OF CONTENTS

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Preparation of Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
ProStain™ Protein Quantification Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Appendix
Section A: Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

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Introduction
Active Motif’s ProStain™ Protein Quantification Kit simplifies protein quantification by providing
highly sensitive detection reagents in a convenient, easy-to-use format. The kit’s detection reagent is also resistant to many effects, which can limit the usefulness of other protein quantification systems, such as pH and many commonly found contaminants; for example, detergents andsalts. In addition, the large Stokes shift, fast reaction time and limited free dye quantum yield
make this kit a significant improvement over other photometric or fluorescent-based detection
systems.
The kit provides lyophilized dye reagent, dilution buffer and BSA for preparation of standards.
Simply resuspend the lyophilized dye in methanol to create the concentrated stock solution, dilute the stock solution, load 100 µl into the wells of a microplate, add 100 µl of sample, mix, thenread the fluorescence. The assay is performed at room temperature, and the signal is stable forup to 2 hours. Common contaminants, such as pH, salts, solvents and some detergents are welltolerated in this assay, but buffers containing high amounts of free amines will affect sensitivity.
Advantages• Large Stokes shift for reduced background• Fast and simple procedure• Robust – limited effect from contaminating substances• Increased quantum yield for improved sensitivity and wide dynamic range

product
ProStain™ Protein Quantification Kit

formatcatalog no.

1000 assays

15001* Sufficient components are provided for performing 1000 assays using fluorescent-based detection. Thisassay can also be easily adapted for use in smaller or larger formats such as 384-well plates or cuvettes.

ProStain™ is for research use only. Not for use in diagnostic procedures.
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Kit Components and Storage
The ProStain Kit is for research use only. Not for use in diagnostic procedures. Kit components
arrive at room temperature. We recommend storing each component at the temperatures
recommended in the table below:
Reagents

Quantity

Storage / Stability

Dye Reagent AM10.5 mg

4°C for 6 months

Dilution Buffer

100 ml

4°C for 6 months

BSA1 mg

4°C for 6 months

Additional materials required• Multi-channel pipettor & pipettor reservoirs• Fluorescent detector• Distilled water• Black microtiter plates or cuvettes• Methanol (for reconstituting the Dye Reagent)

Preparation of Reagents
Dye Reagent Stock Solution
The Dye Reagent is supplied lyophilized. Prepare the Dye Reagent Stock Solution by resuspending the lyophilized Dye Reagent in 12.5 ml methanol in the provided amber bottle. This stocksolution can be stored at 4°C for 6 months.
Dye Reagent Working Solution
Prepare the Dye Reagent Working Solution by diluting the Dye Reagent Stock Solution 1:10 with
distilled water. This solution should be prepared fresh on the day the assay is performed.
Reagent

10 rxns

50 rxns

100 rxns

Dye Reagent Stock Solution

110 µl

550 µl1.1 mlDistilled H2O

990 µl4.95 ml

9.9 mlDilution BufferThis is supplied ready to use.
Stock BSA Solution
The BSA is supplied lyophilized. Prepare the Stock BSA Solution by resuspending the lyophilized
BSA in 1 ml of dH2O in the provided tube to make a 1 mg/ml solution. This stock solution can be
stored at -20°C for 6 months.

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2Protein Quantification Kit Protocol
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
1.

Remove kit contents from 4°C and bring all components to room temperature before use.

2.

Set up a BSA Standard Curve in duplicate using the following concentrations: 10.0, 5.0, 2.5,
1.25, 0.63, 0.32, 0.15 and 0.0 µg/ml. (See table below for suggested layout.)3.

Add 198 µl of Dilution Buffer to wells A1 and A2.

4.

Add 100 µl of Dilution Buffer to wells B1 through H1 and B2 through H2

5.

Pipette 2 µl stock BSA solution (1 mg/ml) into wells A1 and A2.

6.

Mix wells A1 and A2 by pipetting.

7.

Transfer 100 µl from well A1 to B1 and A2 to B2.

8.

Mix wells B1 and B2 by pipetting.

9.

Transfer 100 µl from well B1 to C1 and B2 to C2.

10. Continue this procedure to wells G1 and G2. After mixing, discard 100 µl of solution fromwells G1 and G2.
11.

Wells H1 and H2 are blanks and should contain only 100 µl of Dilution Buffer.

12345678A

10.0 µg/ml

10.0 µg/ml

–

–

–

–

–

–

B

5.0 µg/ml

5.0 µg/ml

–

–

–

–

–

–

C

2.5 µg/ml

2.5 µg/ml

–

–

–

–

–

–

D

1.25 µg/ml

1.25 µg/ml

–

–

–

–

–

–

E

0.63 µg/ml

0.63 µg/ml

–

–

–

–

–

–

F

0.32 µg/ml

0.32 µg/ml

–

–

–

–

–

–

G

0. 15 µg/ml

0. 15 µg/ml

–

–

–

–

–

–

HBlankBlank––

–

–

–

–

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312. Sample wells: For protein determination of unknown samples, prepare a series of dilutions
with the Dilution Buffer, for example: 1:50, 1:100 and 1:200. Pipette 100 µl into each well.
Duplicates of each sample are recommended.
13. Add 100 µl/well of Dye Reagent Working Solution (see Preparation of Reagents section on
page 2 for directions on preparing the Dye Reagent Working Solution) and mix by pipetting
up and down.
14. Incubate for 30 minutes at room temperature (20-25°C) without agitation.
Note:

The signal intensity is stable for up to 1.5 hours after the 30-minute incubation;
read sample before 1.5 hours have elapsed.

15. Measure the fluorescence (excitation: 488 nm, emission: 635 nm).
Note:

When measuring fluorescence, ensure that the gain settings are set to optimal( ~ 140 gain), the number of flashes are set to 3 with no lag time, and theintegration time is ~ 40 µs.

16. Use the standard curve to calculate the protein concentrations of the unknown samples. For
the BSA standard curve, fit with the function y= Ax + B. The r2 should be over 0.95.
Calculation of protein concentration using the BSA standard curve
Average the duplicate readings for each standard and sample and subtract the value obtained
from the zero standard. Plot the fluorescence for the standards against the quantity of thestandards and draw the best fit curve. To quantify the amount of protein in the samples, find thefluorescence value for the samples on the y-axis and extend a horizontal line to the standard
curve. At the intersection point extend a vertical line to the x-axis and read the corresponding
standard value. Note: If the samples have been diluted, the value read from the standard curve
must be multiplied by the dilution factor.

40000
y = 3446.9x + 195.78 r2 = 0.9962

Example standard curve.
The standard curve at rightis provided for demonstration only. You should make astandard curve should be madeevery time an experiment isperformed.

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Emission Intensity (620 nm)

35000
30000
25000
20000
15000
10000
5000
00412

3 4 5 6 7 8Concentration (µg/ml)

910

Appendix
Section A: Troubleshooting Guide
I. Buffer Compatibility and Contaminating Substances
A number of common contaminants have been tested with ProStain, and most are well tolerated;
however, samples containing high concentrations of free amines are not recommended.
ContaminantFinal Concentrationin the assay

Concentration in
100 µl of sample

ResultSodium chloride

20 mM

20 mM

OKSodium chloride

2 mM

4 mM

OKTris

10 mM

20 mM

OKAmmonium sulfate

5 mM

10 mM

OKTween0.001%

0.002%

Not recommended

Triton0.001%0.002%

Not recommended

SDS

0.04%

0.08%

OK

* BSA standards were assayed in the presence or absence of contaminants at the indicated final
concentrations. Equivalent concentrations in 100 µl sample volumes are also listed. This is not acomplete list of contaminants. To determine your buffer compatibility, prepare two BSA curves –one in the same buffer as your samples and one with the supplied Dilution Buffer to determine if
there is any interference.
II. Protein-to-Protein Variation
ProStain determines the protein concentration of a sample relative to a BSA standard curve. If
you are quantitating recombinant or purified protein rather than a protein extract, accuracy may
be improved by using the same protein at a known concentration to make the standard curve, ifavailable.
III. Excitation and Emission Filters
The excitation and emission maxima for Dye Reagent bound to protein are 503 and 602 nm,respectively. We recommend using filters with the following ranges: 485-525 nm Excitation and575-650 nm Emission.

Figure 1: Absorption/emission spectra of free/bounddye.
Normalized absorption and emission spectra of free(solid lines) and conjugated dye (dotted lines) in
phosphate buffer of pH 7.2.
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Fluorescence excitation

absorb (503 nm) emit (600 nm) absorb (612 nm)

1.0UnboundConjugated

0.80.60.4
0.2emit (665 nm)

0400 450 500 550 600 650 700 750 800

Wavelength (nm)Section 4: Technical Services
If you need assistance at any time, please call or send an email to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct:Cell Phone:
E-mail:(86)-21-20926090
18521362870
techchina@activemotif.com

Visit Active Motif online at activemotif.com

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