BRADFORD REAGENT

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).

• Good linear relationship between 10 µg/ml-150 µg/ml protein range

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

Application:

Direct assays of total protein concentration.

Procedures:

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

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).

• Measure the absorbance at 595 nm on a plate reader. 

• 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

Notes:

• Mix the Bradford Reagent thoroughly before use.

• Warm the Bradford Reagent to RT before use.

• 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.

• Measure samples in duplicate to reduce error, plot standard curve every time.

• Measure samples with the lowest concentration first.

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

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