Albumin Blue Fluorescent Assay Kit(version C2)
Catalog No. 15002

Copyright 2007 Active Motif, Inc.

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 isproprietary information and protected by the copyright laws of the United States and international treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2007 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 respectivecompanies.
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TABLE OF CONTENTS

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Preparation of Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Albumin Quantification Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Appendix
Section A. Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

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Introduction
Active Motif’s Albumin Blue Fluorescent Assay Kit simplifies albumin quantification by providing
highly sensitive detection reagents in a convenient and easy to use format. Currently assaysthat are suitable for the determination of low concentrations (<100 mg/L) of albumin in natural
matrices are either nonspecific for albumin or test total protein content such as in dye bindingmethods. In addition, these methods are also complicated and costly. In contrast, the Albumin
Blue Fluorescent Assay Kit is sensitive, specific and easy to use.
The kit provides lyophilized dye reagent, buffers and HSA for preparation of standards. Simply
resuspend the lyophilized dye in isopropanol to create the concentrated stock solution, dilutethe stock solution, load 150 µl into the wells of a microplate, add 25 µl of sample, mix, then readthe fluorescence. The intensity of the fluorescent signal is directly proportional to the albumin
concentration in the sample.1-4 The kit is suitable for use with both individual cuvettes andhigh-throughput 96 well plates.

Advantages• Quick and Easy to Use• Unmatched precision• Sensitive - detection limit (~0.5 mg/L in urine and serum samples)• Highly selective - no interference from other proteins or lipids• High-throughput compatible

product

formatAlbumin Blue Fluorescent Assay Kit250 reactions*catalog no.
15002*Sufficient components are provided for performing 250 assays using fluorescent-based detection. This assay can also beeasily adapted for use in smaller or larger formats such as 384-well plates or cuvettes.

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1Kit Components and Storage
The Albumin Blue Fluorescent Assay 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

QuantityStorage

250 reactions

4°C for 6 months

Buffer A

50 ml

4°C for 6 month

Buffer B

10 ml

4°C for 6 months

HSA

0.2 mg4°C for 6 months

Dye Reagent AM3

Additional Materials Required

P200 and Multi-channel pipettor
Multi-channel pipettor reservoirs
Fluorescent detector
Black microtiter plates or cuvettes
Isopropanol (for reconstituting the Dye Reagent)

Preparation of Reagents
Dye Reagent Stock Solution
The Dye Reagent is supplied lyophilized. Proper reconstitution of the dye is pivotal to the performance of the assay. Prepare the Dye Reagent Stock Solution by resuspending the lyophilized Dye
Reagent in 1 ml isopropanol in the provided amber vial. We recommend using an end-over-end
rotator at room temperature to mix the dye for 30 minutes to ensure complete reconstitution.
Once reconstituted the solution 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 1:50 withBuffer A. This solution should be prepared fresh on the day the assay is performed.
Reagent.

10 rxns

50 rxns

100 rxn

Dye Reagent Stock Soln 33 µl

158 µl

315 µlBuffer A

7.742 ml15.435 ml

1.617 ml

Stock HSA Solution
The HSA is supplied lyophilized. Prepare the Stock HSA Solution by resuspending the lyophilized
HSA in 1 ml of Buffer B in the provided tube to make a 0.2 mg/ml solution. This stock solution canbe 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 HSA Standard Curve in duplicate using the following concentrations: 200, 100,50, 25, 12.5, 6.3, 3.2 and 0.0 µg/ml. (See table below for suggested layout).
Note: We recommend you use a P200 single-channel pipet to set up the standard curve.

3.

Add 50 µl of 200 µg/ml HSA in Buffer B to wells A1 and A2.

4.

Add 25 µl of Buffer B to wells B1 through H1 and B2 through H2

5.

Transfer 25 µl HSA solution (200 µg/ml) from wells A1 and A2 into wells B1 and B2.

6.

Mix wells B1 and B2 by pipetting.

7.

Transfer 25 µl from well B1 to C1 and B2 to C2.

8.

Mix wells C1 and C2 by pipetting.

9.

Transfer 25 µl from well C1 to D1 and C2 to D2.

10. Continue this procedure to wells G1 and G2. After mixing, discard 25 µl of solution fromwells G1 and G2.
11.

Wells H1 and H2 are blanks and should contain only 25 µl of Buffer B.

1

2

3

4

5

6

7

8

9

10

1112A

200µg/ml

200µg/ml

–

–

–

–

–

–

–

–

–

–

B

100
µg/ml

100µg/ml

–

–

–

–

–

–

–

–

–

–

C

50µg/ml

50µg/ml

–

–

–

–

–

–

–

–

–

–

D

25µg/ml

25µg/ml

–

–

–

–

–

–

–

–

–

–

E

12.5
µg/ml

12.5
µg/ml

–

–

–

–

–

–

–

–

–

–

F

6.3
µg/ml

6.3
µg/ml

–

–

–

–

–

–

––––G

3.2
µg/ml

3.2µg/ml––––––––––

HBlankBlank

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312. Sample wells: For albumin determination of unknown samples, prepare a series ofdilutions with Buffer B, for example: 1:50, 1:100 and 1:200. Pipette 25 µl into each well.
Duplicates of each sample are recommended.
NOTE: A clear urine sample is recommended for this assay. If the sample is turbid then it
should be cleared by a centrifugation step.
13. Add 150 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 5 minutes at room temperature (20-25°C) with gentle shaking.
15. Measure the fluorescence (excitation: 560 nm, emission: 620 nm). Best results are obtained
if samples are read within 5 minutes of preparation, however, the signal intensity is stable forup to 30 minutes. (NOTE: When measuring fluorescence, ensure that the gain settings areset to optimal (~ 120-140 gain), the number of flashes are set to 3 with no lag time and thatthe integration time is ~ 40 µs.
16. Use the standard curve to calculate the albumin concentrations of the unknown samples.
For the HSA standard curve, fit with the function y= Ax + B. R2 should be over 0.95.
Calculation of albumin concentration using the HSA standard curve
Average the duplicate readings for each standard and sample. Then subtract the values 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 albumin 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. Alternatively, the equation that best fits the curve can beused to determine the sample concentration.
Example curve:
The following standard curve is provided for demonstration only. A standard curve should bemade every time an experiment is performed.

Emission Intensity (OD620 nm)

45000
y = 196.05x - 676.45 r2 = 0.991

40000
35000
30000
25000
20000
15000
10000
5000
0050

100

150

HSA Standard (µg/ml)
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4200

250

References
1.
2.
3.
4.

Kessler M.A. and Wolfbeis O.S. (1992) Anal. Biochem. 200: 254-259.
Kessler M.A. et al (1992) Clin. Chem. 38(10): 2089-2092.
Kessler M.A. et al (1997) Clin. Chem. 43(6): 996-1002.
Kessler M.A. et al (1997) Anal. Biochem. 248: 180-182.

Appendix
Section A. Troubleshooting
I. Interference by other proteins
Several other proteins were tested for interference at a concentration of 2000 mg/l. Since thefractions of the proteins tested were not essentially free of albumin, part of the response may
have originated from albumin. This implies an even lower interference by these proteins. A
number of common contaminants have been tested with Albumin Blue Fluorescent Assay, andmost are well tolerated; however, samples containing high concentrations of free amines are notrecommended.
ContaminantResponse (%)(human albumin = 100%)

Human transferrin

0.5

Bence Jones protein

0.1

Lactoglobulin

0.8

Lysozyme

0.2

Pepsin0.6

Trypsin

0.2

Bovine γ-globulin

0.2

II. Excitation and Emission Filters
We recommend using filters with the following ranges: 540-580 nm Excitation and 600-640 nm
Emission.

l ores en e ex itation

0.6

0.4

0.2

0500

550

600

650700750

a elengt (nm)Figure 1: Absorption/emission spectra of free/bound dye.
Normalized absorption and emission spectra of free (lower curves) and conjugated dye (upper curves) in theAlbumin Blue Assay.

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5III. Assay limits and recommendations
A clear urine sample is recommended for this assay. If the sample is turbid then it should becleared by a centrifugation step. Although the assay is relatively robust with minor sensitivities topH between 6 and 8 and ionic strength above 200 mmol/L, potential sources of error need to beaddressed. Thus samples with extreme pH, ionic strength or high buffer capacity should be diluted
to warrant constant assay conditions. Evaporation of isopropanol from the microtiter plate well andtemperature changes must be avoided. Best results are obtained if samples are read within 5 minutesof preparation.

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6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
Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com+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

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