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Bradford Protein Assay   

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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. This technique was invented by Bradford (1976).

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.

Acknowledgments

This work was done in the Andrew Binns Lab in the Department of Biology at University of Pennsylvania, USA and supported by National Science Foundation grants MCB 0421885 and IOS-0818613.

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.
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: He, F. (2011). Bradford Protein Assay. Bio-101: e45. DOI: 10.21769/BioProtoc.45.
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If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Sumer Shaykh
Aligarh Muslim university
What is use of phosphoric acid
5/20/2020 4:40:58 PM Reply
Rochana Weerasingha
R Weerasingha
I am preparing to analyze fish larval body protein by Bradford method. Recently I found NaOH is good to prepare protein solution by the sample. But I still have few questions..
1.Is it necessary to add 100 ul water to dye regent when use the blank. If the dye regent is used without 100ul of H2O in blank, is it a issue? Because I already diluted my bio-rad dye regent four times with de-ionized water as per instruction?
2. In protein quantification by UV-1800, do we need use cubic curve instead of linear calibration curve?
3. In the calibration curve, do we need to select the the intersect as zero or not?
4. Actually to prepare 2 ml of protein solution (100 ul will be pipette out as the sample to add with 5 ml dye regent later), how much mg of dry sample or wet sample do we need to take ?
3/15/2020 10:10:11 AM Reply
Tahere Mansour
Babol University of technology
Hello
I have question ,which method is better,Brad ford or lowry ? Why ?
2/19/2020 3:40:00 AM Reply
Guruprasad SA
University of Agriculture sciences Dharwad
which lysis buffer is best for extraction of protein from legume seeds ?
12/1/2019 12:27:44 AM Reply
Hannaneh Rasouli Kenari
Hannaneh Rasouli Kenari
Hi;

What is the difference between coomassie blue r-250 and g-250? Could I use r-250 instead of g-250?
9/23/2019 4:48:17 PM Reply
Theea Becirevic
Faculty of Natural Sciences Sarajevo
Hi,
may I ask for which solutions the Bradford assay can be used, is it suitable for to measure a protein conc. from organic cream?
8/22/2019 10:32:31 AM Reply
MICHAEL SMALLING
Louisiana Tech University

A Bradford's assay is the most common assay for protein concentration. If protein concentration for your sample is completely unknown, you may need to dilute it sufficiently to bring it into the range of your prepared standards. Avoid using SDS or ionic detergents in sample preparation as they may interfere with results.

Mike

8/23/2019 10:31:55 AM Reply


MICHAEL SMALLING
Louisiana Tech University

It also depends on what your "organic cream" is. If it contains a lot of non-protein material, (i.e
Lipids) you may need to extract protein content before assaying using something like trichloroacetic acid to precipitate proteins.

Mike

8/23/2019 10:39:22 AM Reply


Guruprasad SA
University of Agriculture sciences Dharwad
How can I prepare protein sample to determine the protein present in seeds ?
6/18/2019 10:18:50 AM Reply
MICHAEL SMALLING
Louisiana Tech University

Guruprasad, here is a link to an article which should help. https://www.researchgate.net/publication/5236516_Optimizing_protein_extraction_from_plant_tissues_for_enhanced_proteomics_analysis

Mike

6/25/2019 6:47:45 AM Reply


Ronal Luna Pantigoso
universidad nacional de san antonio abad del cusco
Good Morning. under what conditions should the Bradford reagent be stored?
4/27/2019 8:27:27 AM Reply
MICHAEL SMALLING
Louisiana Tech University

Bradford's reagent is not organic and is fine at room temperature for daily use, but should be stored long term at 4-6 C

6/25/2019 6:23:41 AM Reply


abhinav jha
Indian Institute of Science Education and Research Tirupati
What is the role of ethanol in Bradford reagent?
4/21/2019 11:09:09 PM Reply
MICHAEL SMALLING
Louisiana Tech University

Ethanol or methanol, either can be used, is the solvent for your coomassie blue which goes into solution much more readily if you dissolve it in alcohol first. It also helps to prevent some proteins from aggregating during the assay.

6/25/2019 6:26:43 AM Reply


Phuong Tang
Coopers Brewery Ltd.
Hello,
What catalog number of Bovine Serum Abumin (BSA) (Sigma-Aldrich) I need for this assay. Thanks
4/3/2019 3:26:20 PM Reply
MICHAEL SMALLING
Louisiana Tech University

Sigma-Aldrich #05470 is an inexpensive lyophilized powder and will work fine for Bradford's.

4/3/2019 4:52:56 PM Reply


habib afridi
Northeast Normal University
I Dissolved 0.1 g of Coomassie Brilliant Blue G-250 in 50 ml of methanol 95% and added 100 ml 85% (w/v) phosphoric acid (H3PO4), diluted up to 1 liter using distilled water. My question is that when I added water then the colour of comassie brilliant blue G-250 changed from brown to blue. Could anyone tell me that what's the problem. . .
4/1/2019 10:06:00 PM Reply
MICHAEL SMALLING
Louisiana Tech University

Habib, it sounds like your DI water may be acidic.

4/2/2019 5:31:59 AM Reply


Ronal Luna Pantigoso
universidad nacional de san antonio abad del cusco
hello, what is the role of methanol and phosphoric acid in the bradford reagent. Thank you
2/4/2019 11:31:16 AM Reply
Eva Steinmetz
Saarland University

This Assay is done with blue acid dye. The phosphoric acid generates the acidic environment. This (and beeing bound to protein) is needed to shift of the acid dye (e.g. coomassie G250) absorbtion maximum from 465 nm to 595 nm . After that, you are able to measure the protein´s concentration at 595 nm.

2/5/2019 1:54:01 AM Reply


anu mrithi
anna university
what is the difference between bsa assay and bradford assay
12/12/2018 7:33:10 PM Reply
MICHAEL SMALLING
Louisiana Tech University

bsa assay simply refers to the use of bsa as a standard for the procedure, and could mean a Bradford's or BCA assay, which uses bicinchoninic acid.

12/22/2018 7:07:32 AM Reply


jasim al-sede
university of Bucharest
How to prepare BSA 1mM by using 0.660 g
11/22/2017 9:47:43 AM Reply
MICHAEL SMALLING
Louisiana Tech University

BSA can vary, but is generally calculated using 66kDa as a standard weight. So 66g/L. or 0.066g/mL is 1.0mM concentration. 0.660g is then sufficient for 10mL at 1.0mM concentration.

12/22/2018 7:13:50 AM Reply


jasim al-sede
university of Bucharest
Hello, I am a new subscriber jasim
11/22/2017 9:43:14 AM Reply
Sneha More
Ph.D. student
hi every one , i am facing problem is plotting std protein graph. here i used 1mg/ml std protein. different concentration used for plotting is 0.2mg / 4ml 0. 4mg/ 4ml 0.6mg/ 4ml 0.8 mg/ 4ml and 1mg /4ml. and for estimation of protein only 50 ul sample was used form each above concentration for protein estimation than 2.5 ml of Bradford reagent was added to each tube and read at 595.. what concentration of protein is should take on X axis to plot a std protein graph. is it 0.2mg / 4ml 0. 4mg/ 4ml 0.6mg/ 4ml 0.8 mg/ 4ml 1mg /4ml. or according to 50 ul what ever protein concentration will be, that i need to used on X axis ? please suggest. i need to plot a std graph of protein but i followed above protocol, please help
10/20/2017 2:47:35 AM Reply
MICHAEL SMALLING
Louisiana Tech University

Hi Sneha, be advised that you are using very low concentrations for your Bradford's assay standards. Typically, you would use those amounts per 1mL rather than 4mL. However, as you mix your unknown samples at the same rate as your standards, 50uL/2.5mL, the readings for your standard will correlate to the same protein concentration in your unknown samples.

12/22/2018 7:23:39 AM Reply


mahmoud ali
agriculture research center egypt
Bradford reagent
could i use it after preparation or must incubate over night before using
the volume of Bradford reagent 1.5 ml and 30 µl for sample ???
2/14/2017 9:25:12 AM Reply
MICHAEL SMALLING
Louisiana Tech University

Mahmoud, the recommendation for incubation overnight is to allow complete dissolution of your dry reagents. If you are confident that your reagents are all thoroughly dissolved, you can use it immediately. Most formulations of a Bradford's reagent use a protein/reagent ratio of 1:25 (10uL/250uL; 100uL/2.5mL). Hope this helps, Mike

12/22/2018 7:50:56 AM Reply


mahmoud ali
agriculture research center egypt
can i replace phosphoric with orthophosphoric acid?
and if i can could i use it without dilution direct from bottle
2/11/2017 1:55:15 AM Reply
Fanglian He
Bio-protocol

Hi,

Phosphoric acid and orthophosphoric acid are often considered as the same thing (http://www.sigmaaldrich.com/catalog/product/aldrich/345245?lang=en®ion=US).

The one mentioned in the protocol was 85% (w/v) from vendor. I did not dilute it. Whatever the concentration of your H3PO4 stock is, its final concentration in the reagent should be 8.5% (w/v).

Hope this helps.
Fanglian


2/14/2017 12:51:57 AM Reply


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.
10/19/2016 6:41:36 PM Reply
Fanglian He
Bio-protocol

Hi Le,

Sorry to miss your questions. Yes, you were right that we should adjust the final vol.. As 50mg Coomassie G250 would not change the vol significantly, I did not write it in that way.

That is good point. I never got chance to try Ethanol. Please share your experience here if it works in your hand.

Thanks,
Fanglian

2/14/2017 12:57:35 AM Reply


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.
10/19/2016 6:41:35 PM Reply
Raj Kumar
JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY

Hi! You can use Ethanol instead of Methanol.

12/7/2017 10:20:13 AM Reply


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?
4/15/2015 12:19:07 PM Reply
Fanglian He
Department of Biology, University of Pennsylvania, USA

Hi,

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

Good luck,
Fanglian

4/17/2015 12:03:06 AM Reply


Wynifred Wang
China Aguriculral University(CAU,China)
Vary Good.
12/5/2013 7:11:38 AM Reply
Alaa Elminisy
National research center

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

12/9/2015 12:21:48 AM Reply


Fanglian He
Bio-protocol

Hi Alaa,

It is 1x.

2/14/2017 1:02:08 AM Reply


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