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
Equipment
Procedure
Recipes
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
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Volume of protein standard(ml) * starting protein Concentration = Amount of protein(mg)
kahan se copy kea Baji?
Sorry ??
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
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
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_analysisMike
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
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.
Sigma-Aldrich #05470 is an inexpensive lyophilized powder and will work fine for Bradford's.
Habib, it sounds like your DI water may be acidic.
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.
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.
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.
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.
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
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
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
Hi! You can use Ethanol instead of Methanol.
Hi,Yes, it could cause the problem. You may try BCA protein assay instead.Good luck,Fanglian
Hi dears, Bradford reagent used diluted(1x) or 5x??
Hi Alaa,It is 1x.
