When measuring protein concentration with a spectrophotometer, is the use of Coomassie Brilliant Blue necessary? Protein (BSA) has a peak in UV, so can I prepare different concentrations of it and determine the unknown concentration?
Dear Sayed,
For determining protein concentration using spectrophotometer you don't need to use Coomassie Brilliant Blue.
You calculate the protein concentration by measuring its absorbance at 280 nm. If you know the molar extinction coefficient of your protein, then by using beer-lambert law you can calculate the absolute concentration of your sample. You don't even need a standard curve. And if you know the amino acid composition of your protein, you can easily calculate its molar extinction coefficient (it depends upon amino acids W, Y and F). You can calculate molar extinction coefficient here: http://web.expasy.org/protparam/
The only thing you need to be careful about is that when measuring absorbance of your sample at 280 nm, you must be in the linear range of your spectrophotometer (generally for most of the instruments this range is from 0.1 to 1.0 O.D.).
I hope it will help you.
Best,
Jogender
Coomassie Brilliant Blue is generally used for staining of proteins in a polyacrylamide gel.
Although Coomassie Brilliant Blue is also used in bradford assay which is used to determine protein concentration, protein concentration determination by absorbance at 280 nm wavelength is fairly simple and robust and it's a non-invasive method.
Regards,
Jogender
You could use the Bradford method, which is based on Coomassie Blue binding to protein, with absorbance change at 595 nm. BSA is often used as a standard.
Alternatively, you could use the approximately 280 nm absorbance of the protein (with no dye), as explained by Jogender Singh. If you do this, I recommend denaturing your protein fully using 8M guanidine HCl. Then you can use the calculated extinction coefficient method. Otherwise, turbidity caused by protein aggregation tends to elevate the UV absorbance. The UV method should only be used with highly purified proteins.
Similar problems faced me. I want to determine the SOD, T-AOC and MDA antioxidative activities of chicken intestinal mucosal tissues. Before testing samples for OD values for the above parameters, I must first test the protein concentration. Which method is best? I will use a spectrophotometer (microplate reader) to test the samples for OD values. Thank you so much.
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