1Rat Urinary Protein Assay Kit
Catalog # 9040
For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

Assay kit to evaluate urinary protein concentration

FORMAT:

96-well ELISA Plate with removeable strips

ASSAY TYPE:

Turbidity Assay

ASSAY TIME:

10 minutes

STANDARD RANGE:

4 mg/ml to 0.4 mg/ml

NUMBER OF SAMPLES:

Up to 16 (duplicate) samples/plate

SAMPLE TYPES:

Urine

RECOMMENDED SAMPLE DILUTIONS:

1:2 (at least)

CHROMOGEN:

N/A (read at 450 nm)

STORAGE:-20°C for 12 months

VALIDATION DATA:

N/ANOTES:

N/A© 2023 Chondrex, Inc. All Rights Reserved, 9040 4.1
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

2

Rat Urinary Protein Assay Kit
Catalog # 9040
For Research Use Only - Not Human or Therapeutic Use

INTRODUCTION
The turbidity assay method (1) has been widely used to determine urinary protein levels in human specimens because it is accurate, easy,and economical. However, urine volume collected from rats varies from 0.1 ml to 20 ml during a 16-hour collection period, thus the volumeis occasionally insufficient for current assay methods. In addition, turbidity (OD 450 nm) readings of individual test tubes using aspectrophotometer may be cumbersome. Chondrex, Inc.’s Rat Urinary Protein Assay Kit (Cat # 9040) uses the turbidity method in 96-wellplates, offering a solution for assaying a large number of rat urine samples.
Protein concentrations in urine samples can be determined by turbidity or Bradford assay methods (2). The turbidity assay method whichutilizes 3% sulfosalicylic acid is more convenient than the Bradford assay method for assaying a large number of samples because of thewide range of the dose response curve (Ex: 0.4 to 4 mg/ml) and the stable turbidity. Regardless of the assay method used, bovine serumalbumin (BSA) cannot be used as a standard. For example, in the turbidity assay, the dose response curve generated by BSA significantly
differs from that of serum proteins. In the Bradford assay, OD value of globulins is only 70% that of BSA (3). Therefore, a standard proteinsolution prepared from normal rat serum is ideal for assaying urinary protein levels instead of using BSA.
NOTE: The Bradford assay method requires two separate regression curves for assaying protein concentration, from 0.05 to 0.6 mg/ml andfrom 0.5 to 1.5 mg/ml. Because the protein concentration in rat urine can vary from 0.1 mg/ml to 50 mg/ml depending upon the progress ofnephritis, various dilutions of individual samples are required.

KIT COMPONENTS
Item

Quantity

AmountStorage

Standard Protein Solution (90401)
PBS (90402)

1 vial
1 bottle

1.5 ml, 4 mg/ml
30 ml

–20°C
Room Temperature

0.1N HCl (90403)
3% Sulfosalicylic Acid (90404)
96-well Plate

1 bottle
1 bottle
2 plates

40 ml
40 ml
96-well (8-well strips x 12)

Room Temperature
Room Temperature
Room Temperature

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

Add 250 µl of 3% Sulfosalicylic acid to wells (0.1N HCl for blank wells)
Incubate at room temperature for 10 minutes

Read at 450 nm© 2023 Chondrex, Inc. All Rights Reserved, 9040 4.1
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

URINE COLLECTION PROTOCOL
Collect urine from 5 pm to 9 am every other day with metabolic cages. Measure the urine volume and centrifuge to remove insoluble materials.
Keep the supernatant in a refrigerator for short-term storage and at –20°C for long-term storage.

PLATE LAYOUT
The assay should be performed in duplicate for both standards and samples. Because urine samples and even the standard may be colored
due to hemoglobin contamination, it is important to determine the individual background values and subtract them from the turbidity values
in sample wells. Therefore, columns 3-4, 7-8, and 11-12 are used as blank wells.
123456789101112A

44SB4

SB4

S1S1

SB1

SB1S9S9SB9SB9B

3.2

3.2

SB
3.2

SB
3.2S2

S2SB2

SB2

S10

S10

SB10

SB10

C

2.4

2.4

SB
2.4

SB
2.4S3S3

SB3

SB3

S11

S11

SB11

SB11

D

1.6

1.6

SB
1.6

SB
1.6S4

SB4

SB4

SB4

S12S12SB12SB12E

1.2

1.2

SB1.2

SB
1.2

S5S5SB5

SB5S13S13

SB13

SB13

F0.80.8

SB0.8

SB0.8

S6S6SB6SB6S14S14SB14

SB14

G0.40.4

SB0.4

SB0.4

S7S7

SB7

SB7

S15

S15

SB15

SB15HB

B

SB

SB

S8

S8

SB8

SB8

S16S16

SB16

SB16StandardsSamples

NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and fordisposal.

ASSAY PROCEDURE1.

Prepare Standards: Add 0, 5, 10, 15, 20, 30, 40, and 50 µl of Standard Protein Solution (4 mg/ml) in duplicate into columns 1-2(standard wells) and 3-4 (standard blank wells) from rows A to H according to the plate mapping layout. Add PBS to individual wells toadjust the final volume to 50 µl.
Standard (µl/well)

05101520304050

PBS (µl/well)

50

4540

353020100

Total Volume (µl)

5050505050505050

Final Concentration (mg/ml)

00.4

0.8

1.2

1.6

2.4

3.2

4.0© 2023 Chondrex, Inc. All Rights Reserved, 9040 4.1
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

42.

Prepare Samples: Centrifuge urine samples at 10,000 rpm for 3 minutes using a tabletop microcentrifuge. Add 1-50 µl of urine
supernatant in duplicate into columns 5-6 (test samples) and 7-8 (sample blanks) from rows A-H and into columns 9-10 (test samples)and 11-12 (blanks) from rows A-H according to the plate mapping layout. Add PBS to adjust the sample volume to 50 µl. Alternatively,
dilute samples 1:2-1:50 or more with PBS and add 50 µl of diluted sample per well.

3.

Add Reagents: Add 250 µl of 3% sulfosalicylic acid into standard and sample columns 1-2, 5-6, and 9-10, and 250 µl of 0.1N HCl into
blank columns 3-4, 7-8, and 11-12. Incubate the plate for 10 minutes at room temperature and then read plate using a plate reader atOD 450 nm (single beam).

CALCULATING RESULTS
1.

Average the duplicate OD values for the blank, standards, test samples, and sample blanks.

2.

Subtract the averaged blank OD value from the individual averaged standard, standard blank, sample, and sample blank OD values.
Subtract the averaged standard blank and sample blank OD values from their corresponding averaged standard and test sample OD
values.

3.

Plot the adjusted OD values of the standards against the concentration (mg/ml) of standard protein. Figure 1 shows representative OD
values of the standard range from 0.4 - 4 mg/ml.

4.

The protein levels in samples can be calculated using regression analysis. If necessary, multiply it by the sample dilution factor toobtain the urinary protein concentration in the original sample. For additional assistance, please download a sample calculation
worksheet from www.chondrex.com. Copy the raw data into the template’s cells C9-N16, redo the trendline and display the newtrendline equation. Then type the “a” and “b” values from the equation of the standard regression curve into cells M38 and M39
respectively. Type in the volumes used for the samples (cells D60-D75) and PBS (cells E60-E75) to calculate sample dilution. The
protein concentration in urine will be displayed in the last column (cells N60-N75).
Determining the total amount of protein secreted in urine is preferred over the urinary protein concentration alone because the protein
concentration is generally high when urine volume is small. Chondrex, Inc. recommends determining the total amount of protein
secreted in urine in 16- or 24-hour periods to determine renal function.
Total urinary protein (mg) = concentration (mg/ml) x volume of urine (ml)Figure 1 - A Typical Standard Curve for the Rat Urinary Protein Assay Kit© 2023 Chondrex, Inc. All Rights Reserved, 9040 4.1
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

5

VALIDATION DATA
Seventy-two urine samples collected from normal and nephritic rats were assayed for their protein concentration using both the turbidity andthe Bradford assay methods. Using an identical standard prepared from normal rat serum, the resulting data was analyzed for correlation byregression analysis. As shown in Figure 2, the protein concentration determined by both methods correlated well with a correlation coefficientof r2 = 0.928.
Figure 2 - Correlation of Urinary Protein Levels Determined by the Turbidity and Bradford Assay Methods. For assaying protein concentration
with the turbidity method, 50 µl of urine from normal rats and 10 µl from nephritic rats were used. Similarly, 10 µl from normal rats and 2.5µl from nephritic rats were used for the Bradford method. In the Bradford method, 13 out of 72 samples were outside of the assay range due
to high protein concentration. However, using the turbidity method, only 3 samples were out of assay range and needed to be re-assayedwith further dilution.

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

REFERENCES
1.

H. Nishi, R. Elin, Three turbidimetric methods for determining total protein compared. Clin Chem 31, 1377-80 (1985).

2.

M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72, 248-54 (1976).

3.

Bio-Rad Protein Assay. Bio-Rad Laboratories, California, USA© 2023 Chondrex, Inc. All Rights Reserved, 9040 4.1
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