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The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex. Unlike the BCA assay, reducing agents (i.e. DTT and beta—mercaptoethanol) and metal chelators (i.e. EDTA, EGTA) at low concentration do not cause interference. However, the presence of SDS even at low concentrations can interfere with protein-dye binding.

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[Bio101] Bradford Protein Assay

Biochemistry > Protein > Quantification
Author: Fanglian He
3/20/2011, 71417 views, 4 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.45

[Abstract] The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the basic amino acid residues, arginine, lysine and histidine, which contributes to formation of the protein-dye complex. Unlike the BCA assay, reducing agents (i.e. DTT and beta—mercaptoethanol) and metal chelators (i.e. EDTA, EGTA) at low concentration do not cause interference. However, the presence of SDS even at low concentrations can interfere with protein-dye binding.

Materials and Reagents

  1. Bovine Serum Abumin (BSA) (Sigma-Aldrich)
  2. Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number: 27815)
  3. Methanol
  4. Phosphoric acid (H3PO4)
  5. Bradford reagent (see Recipes)

Equipment

  1. Spectrophotometer (Tecan)
  2. Whatman #1 paper (Whatman)

Procedure

  1. Standard assay procedure (for sample with 5-100 µg ml-1 protein)
    1. Prepare five to eight dilutions of a protein (usually BSA) standard with a range of 5 to 100 µg protein.
    2. Dilute unknown protein samples to obtain 5-100 µg protein/30 µl.
    3. Add 30 µl each of standard solution or unknown protein sample to an appropriately labeled test tube.
    4. Set two blank tubes. For the standard curve, add 30 µl H2O instead of the standard solution. For the unknown protein samples, add 30 µl protein preparation buffer instead. Protein solutions are normally assayed in duplicate or triplicate.
    5. Add 1.5 ml of Bradford reagent to each tube and mix well.
    6. Incubate at room temperature (RT) for at least 5 min. Absorbance will increase over time; samples should incubate at RT for no more than 1 h.
    7. Measure absorbance at 595 nm.

  2. Microassay procedure (<50 µg ml-1 protein):
    1. Prepare five standard solutions (1 ml each) containing 0, 10, 20, 30, 40 and 50 µg ml-1 BSA
    2. Pipet 800 μl of each standard and sample solution (containing for <50 µg ml-1 protein) into a clean, dry test tube. Protein solutions are normally assayed in duplicate or triplicate.
    3. Add 200 μl of dye reagent concentrate to each tube and vortex.
    4. Follow the procedure described above for the standard assay procedure.

Recipes

  1. Bradford reagent
    Dissolve 50 mg of Coomassie Brilliant Blue G-250 in 50 ml of methanol and add 100 ml 85% (w/v) phosphoric acid (H3PO4).
    Add the acid solution mixture slowly into 850 ml of H2O and let the dye dissolve completely (note: Do not add H2O into the acid solution).
    Filter using Whatman #1 paper to remove the precipitates just before use.
    Store in a dark bottle at 4 °C.

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Stoscheck, C. M. (1990). Quantitation of protein. Methods Enzymol 182: 50-68.


How to cite this protocol: He, F. (2011). Bradford Protein Assay. Bio-protocol Bio101: e45. DOI: 10.21769/BioProtoc.45; Full Text



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10/19/2016 6:41:36 PM  

Le Nghia
Laboratory of Molecular Biotechnology

In the dye reagent's recipe, I wonder how much is the total volume of reagent. Is it about 150 ml, include 50 mg Coomassie G250, 50 ml Methanol, and 100 ml 85% Phosphoric acid? Don't we need to add mQ to adjust final volume?
And I think Methanol is poisonous, so can I replace it by Ethanol?
Thanks.

Reply

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10/19/2016 6:41:35 PM  

Le Nghia
Laboratory of Molecular Biotechnology

In the dye reagent's recipe, I wonder how much is the total volume of reagent. Is it about 150 ml, include 50 mg Coomassie G250, 50 ml Methanol, and 100 ml 85% Phosphoric acid? Don't we need to add mQ to adjust final volume?
And I think Methanol is poisonous, so can I replace it by Ethanol?
Thanks.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

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4/15/2015 12:19:07 PM  

nebula PH
gorgan university of medical science

hi dears
im using SDS in my lysis buffer so it could make problem for my bradford assay?

4/17/2015 12:03:06 AM  

Fanglian He (Author)
Stanford

Hi,

Yes, it could cause the problem. You may try BCA protein assay instead.

Good luck,
Fanglian

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12/5/2013 7:11:38 AM  

Wynifred Wang
China Aguriculral University(CAU,China)

Vary Good.

12/9/2015 12:21:48 AM  

Alaa Elminisy
National research center

Hi dears,
Bradford reagent used diluted(1x) or 5x??

Reply

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