1Bromophenol Blue Protein Assay Kit
Catalog # 6026

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to quantify proteins (proteinuria)

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Colorimetric Assay

ASSAY TIME:

5 minutes

STANDARD RANGE:

2 mg/ml to 0.03 mg/ml

NUMBER OF SAMPLES:

Up to 40 (duplicate) samples/plate

SAMPLE TYPES:

Urine, Serum, and Plasma

RECOMMENDED SAMPLE DILUTIONS:

VariesCHROMOGEN:

N/A (read at 610 nm)

STORAGE:

4°C

VALIDATION DATA:

Intra-Assay (1.7-5.2%)/Inter-Assay (4.4-9.8%)/Spiking Test (93-105%)

NOTES:© 2020 Chondrex, Inc. All Rights Reserved, 6026 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2Bromophenol Blue Protein Assay Kit
Catalog # 6026

For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
Bromophenol blue (BPB), a phenolphthalein anionic dye, binds to proteins under neutral to acidic conditions. From this attribute, BPB candetermine protein levels in samples, especially in solubilized cultured cells and cell membrane proteins with high concentrations of surfactants(8). This is an advantage over the widely used Coomassie blue protein assay which is affected by the presence of surfactants in the samples.
Proteinuria, the presence of an excess of serum proteins in the urine, is a useful marker of renal disease. In addition, the pathophysiology ofproteinuria can be divided into tubular or glomerular dysfunction. For example, albumin, a plasma protein, may better define pathologicalproteinuria of glomerular origin while beta 2-microglobulin indicates tubular origin (6, 7). Usually, urinary protein levels are determined using
a dipstick assay. However, this method is affected by urine volume and color, which can lead to inaccurate results (1-3). Therefore, as asimple, precise, and accurate alternative for quantifying proteinuria, a BPB protein assay is available. The results may better reflect
glomerular albuminuria due to its higher affinity for albumin rather than globulin (4, 5).
Chondrex, Inc. provides a BPB Protein Assay Kit (Cat # 6026) to evaluate proteinuria in mice and rats, as well as a Rat Urinary Protein Assay
Kit (Catalog # 9040), a Rat Albumin Detection ELISA Kit (Catalog # 3020), and a Mouse Albumin Detection ELISA Kit (Catalog # 3012).

KIT COMPONENTS
Item

Quantity

AmountStorage

Bovine Serum Albumin Standard (60261)

1 vial2 mg, Lyophilized

4°C

Solution A (60262)
Solution B (60263)
PBS (60264)
ELISA Plate

1 bottle
1 bottle
1 bottle
1 each

18 ml
6 ml
50 ml

4°C
4°C
4°C
4°C

96-well (8-well strips x 12)

ASSAY OUTLINE
Add 50 µl of diluted standards and samples to wells

Add 200 µl of dye solution to wells

Incubate at room temperaturefor 5 minutes

Read at 610 nm© 2020 Chondrex, Inc. All Rights Reserved, 6026 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3

PLATE LAYOUT
123

4

5

6

7

8910

1112A

22

S1

S1

S9S9

S17

S17

S25

S25

S33

S33

B

11S2

S2

S10

S10

S18

S18

S26

S26

S34

S34C0.50.5

S3S3

S11

S11

S19

S19

S27

S27

S35

S35

D0.25

0.25

S4S4

S12

S12

S20

S20

S28

S28

S36

S36

E

0.125

0.125

S5S5S13

S13

S21

S21

S29

S29

S37

S37

F

0.63

0.63

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G

0.31

0.31

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

HB

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

Samples

NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Crystals may form in Wash Buffer, 20X when stored at cold temperatures. If crystals have formed, warm the wash buffer by placing
the bottle in warm water until crystals are completely dissolved.
NOTE 4: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 5: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and fordisposal.

ASSAY PROCEDURE
1.

Prepare Standard Dilutions: The recommended standard range is 0.031 - 2 mg/ml. Dissolve one vial of Standard in 1 ml of PBS forthe 2 mg/ml standard and serially dilute with PBS. For example, mix 200 µl of the standard (2 mg/ml) with an equal volume of PBS tomake a 1 mg/ml solution, and then repeat it five more times for 0.5, 0.25, 0.125, 0.063, and 0.031 mg/ml solutions. The remaining 2mg/ml standard stock may be stored at 4°C for future use. Chondrex, Inc. recommends making fresh serial dilutions for each assay.
1000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml23456781000 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml

250 ml2 mg/ml

1 mg/ml

0.5 mg/ml

0.25 mg/ml

0.125 mg/ml

0.063 mg/ml

0.031 mg/ml

Blank

1

© 2020 Chondrex, Inc. All Rights Reserved, 6026 2.016928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

42.

Prepare Sample Dilutions: Dilute samples with PBS. It is recommended to use 2-3 different dilutions if the sample albumin level isunknown.

3.

Prepare Dye Solution: Mix 150 µl of Solution A with 50 µl of Solution B for each well just before use. For example, 8 samples, 7-pointstandards, and one blank (all in duplicate) will require 6.4 ml of the Dye Solution. Mix 4.8 ml of Solution A with 1.6 ml of Solution B.

4.

Add Standards and Samples: Add 50 µl of standards, PBS (Blank), and samples to designated wells.

5.

Add Dye Solution: Add 200 µl of the dye solution into all wells. Incubate at room temperature for 5 minutes.

6.

Read Plate: Read the OD values at 610 nm. If the OD values of samples are greater than the OD values of the highest standard, reassay the samples at a higher dilution.

CALCULATING RESULTS
1.

Average the duplicate OD values for the standards, blank (B), and test samples.

2.

Subtract the “blank” (B) values from the averaged OD values of the standards and test samples.

3.

Plot the OD values of standards against the mg/ml of standard. Using a log/log plot will linearize the data. Figure 1 shows a
representative experiment where the standard range is from 0.031-2 mg/ml.

4.

The mg/ml of protein in test samples can be calculated using regression analysis. Multiply it by the sample dilution factor to obtain theprotein concentration (mg/ml) in original test samples. For additional assistance, please download a sample calculation worksheet fromwww.chondrex.com.

Figure 1 - A Typical Standard Curve for the BPB Assay Kit
10.00
R² = 0.9963

OD 610 nm

1.00

0.10

0.01
0.01

0.1
1Protein Concentration (mg/ml)

10© 2020 Chondrex, Inc. All Rights Reserved, 6026 2.016928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

5VALIDATION DATA
Table 1 - Reproducibility Data for the BPB Assay Kit
Test At

0.07 mg/ml

0.3 mg/ml

1 mg/ml

Intra-Assay CV (%)

1.7

5.2

3.3

Inter-Assay CV (%)

9.8

6.2

4.4

Spiking Test*
105%
101 %
93 %
*Standard was added with known amounts of albumin and then diluted with PBS to assay the protein concentration.

TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s Assay FAQ for more information.

REFERENCES
1.

D. Ellis, G. Buffone, New Approach to Evaluation of Proteinuric States. Clin Chem 23, 666-70 (1977).

2.

P. Peterson, P. Evrin, I. Berggård, Differentiation of glomerular, tubular, and normal proteinuria: determinations of urinary excretion of
2-microglobulin, albumin, and total protein. J Clin Invest 48, 1189-1198 (1969).

3.

W. Gyure, Comparison of Several Methods for Semiquantitative Determination of Urinary Protein. Clin Chem 23, 876-9 (1977).

4.

B. Garner, C. Wiedmeyer, Comparison of a Semiquantitative Point-Of-Care Assay for the Detection of Canine Microalbuminuria With
Routine Semiquantitative Methods for Proteinuria. Vet Clin Pathol 36, 240-4 (2007).

5.

L. Killingsworth, Clinical Applications of Protein Determinations in Biological Fluids Other Than Blood. Clin Chem 28, 1093-102 (1982).

6.

K. Schosinsky, M. Vargas, A. Luz Esquivel, M. A. Chavarria, Simple spectrophotometric determination of urinary albumin by dye-binding
with use of bromophenol blue. Clin Chem 33, 223-226 (1987).

7.

R. Flores, A Rapid and Reproducible Assay for Quantitative Estimation of Proteins Using Bromophenol Blue. Anal Biochem 88, 605-11(1978).

8.

C. Greenberg, P. Craddock, Rapid Single-Step Membrane Protein Assay. Clin Chem 28, 1725-6 (1982).

© 2020 Chondrex, Inc. All Rights Reserved, 6026 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com