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Bradford Assay

Method

Standard

200 µg/ml
BSA Stock

Final Protein Concentration

Wells

1

0 µl

0 µg/200 µl

A1,B1

2

2 µl

0.4 µg/200 µl

C1, D1

3

3 µl

0.6 µg/200 µl

E1, F1

4

4 µl

0.8 µg/200 µl

G1, H1

5

5 µl

1.0 µg/200 µl

A2, B2

6

7.5 µl

1.5 µg/200 µl

C2, D2

7

10 µl

2.0 µg/200 µl

E2, F2

8

12.5 µl

2.5 µg/200 µl

G2, H2

 

Bring to 10 µl with dH2O.

Add protein to 200 µl Bradford dye solution to final concentrations listed above for each standard.

Enter these numbers into plate counter.

  1. Prepare in a 96-well plate for reading in a plate reader.
  2. All samples and standards are prepared in duplicate.
  3. For each standard -- except for standard 8 -- add enough water to bring to 10 µl.

  4. Dilute samples 1:10 or 1:100 in water in duplicate and load 10 µl per lane in row 3.
  5. Dilute 1 part Bradford Reagent to 4 parts dH2O and mix well.
  6. Be sure to make more than enough dilute reagent for all the samples.

    If you run out of reagent part way through the plate, you will have to discard everything with the old reagent and start over. Samples done with a different dye dilution than your standards will not be accurate.

  7. Add 200 µl dilute Bradford reagent to each well. Use the dye soon after diluting and read the plate immediately.

    The reagent is only good for ~20 minutes.

  8. After the plate has been read, make sure your standards are linear. If not, try reading it one more time. Otherwise, you will have to start over from the beginning.
  9. Determine concentration:

    10 µl of a 1:100 dilution = [(concentration as read *100)/10] µg/µl

© 2014 The University of Texas MD Anderson Cancer Center