Introduction:

Bradford method utilizes Coomassie Brillant Blue G-250 dye binding to an unknown protein and

forming a complex which can be detected spectophotometrically at 595 nm.

Features:

Rapid and economical: entire procedure (10-20 samples) only takes about 10 minutes (timefor preparation of samples is not included).

High sensitivity: the minimum protein quantity which could be tested is 0.2 µg in 1-20 µl volume.

Direct assays of total protein concentration.

Protocols for Measurements in Test Tubes A. Make Dilutions of BSA standards and Sample of interest.

Label 8 test tubes from #1 to #8. Make BSA serial dilutions as follows:

In Tube #1, add 20 µl of original BSA protein standard (5 mg/ml) + 780 µl of PBS.

In Tube #2, transfer 100 µl of PBS + 400 µl from Tube #1.

In Tube #3, transfer 100 µl of PBS + 400 µl from Tube #2.

In Tube #4, transfer 120 µl of PBS + 360 µl from Tube #3.

In Tube #5, transfer 120 µl of PBS + 240 µl from Tube #4.

In Tube #6, transfer 150 µl of PBS + 150 µl from Tube #5.

In Tube #7, transfer 100 µl of PBS + 100 µl from Tube #6.

In Tube #8, transfer 100 µl of PBS ONLY.

This is BSA standard serial dilution

	1	2	3	4	5	6	7	8
BSA final concentration (μg/ml)	125	100	80	60	40	20	10	0

Now dilute sample of interest to preferred concentration (10-125 μg/ml). Record dilution factor X.

Label Tubes #9 to #11

In Test Tube #9, transfer 20 μl of diluted protein sample of interest.

In Tube #10, transfer 20 μl of diluted protein sample of interest, preferred at a different dilution factor.

In Tube #11, transfer 20 μl of PBS ONLY.

B. Measure concentration of BSA standards and sample of interest

#1 to #8 to each well in a 96-well plate. Make duplicates as a good laboratory practice. Add 200µl of Bradford Reagent to each tube. Mix with the plate shaker for 30 seconds and incubate plate for 10 minutes at room temperature (RT).

Subtract the average 595 nm absorbance measurement of the Blank standard replicates from the 595 nm absorbance measurement of all other individual standard and unknown sample replicates.

Plot standard curve using the average Blank-corrected 595 nm measurement for each BSA standard vs. its concentration in μg/ml.

Using BSA protein standard curve, calculate concentration of diluted protein sample and original protein concentration with dilution factor X

If there is detergent present in the sample, please use BCA Protein Assay Kit (SK3021, SK3051).

Absorbance of protein varies; the results will be more accurate if the protein of interest were used to obtain the standard curve.

PRODUCTS ARE INTENDED FOR BASIC SCIENTIFIC RESEARCH ONLY!
NOT INTENDED FOR HUMAN OR ANIMAL USE!

Disclaimer: For laboratory research use only.