I am using Bradford Assay to determine the unknown concentration of a protein. I have used BSA standard solutions to get a standard curve and then use it to determine the concentration of my unknown sample.
However, since the two proteins are different, to calculate the molarity of the solution, what molecular weight should I use? I believe the extinction coefficient will be different from that obtained from the standard curve, since the proteins are different.
The accuracy of the Bradford assay depends on the similarity in amino acid composition (not sequence) between the sample protein and the standard. If your protein has a very different amino acid composition from BSA, there will likely be a systematic error in the measurement leading to an overestimate or underestimate of the concentration.
The Bradford assay is typically read out in micrograms of protein. Divide that by the volume of the sample used for the test to get µg/µL (= mg/mL = g/L). Then, divide that by the molecular mass of the protein (g/mole) to get the concentration in mol/L.
The extinction coefficient of the protein, used for a concentration measurement by UV absorbance, is irrelevant for the Bradford assay. If you choose to measure the protein concentration by UV absorbance, the extinction coefficient can be calculated from the amino acid composition according to the method of Gill and von Hippel.
Article Calculation of Protein Extinction Coefficients from Amino Ac...
This measurement must be made in 6 M guanidine-HCl to be accurate. This method is only useful for essentially pure proteins without bound ligands or prosthetic groups.
The Lowry and BCA protein assays are less affected by the amino acid composition of the protein than the Bradford assay, if you are concerned about the selection of the appropriate standard protein.
BSA is unusual in having hydrophobic pockets (for hormone binding), which bind more CBB than "average" proteins. Unless your protein of interest also has such pockets, immunoglobulin is a better standard to use for the Bradford assay.
In general, all protein determination methods give relative results. That is, all you can say is that you get the same absorbance as x µg of standard protein, but this doesn't necessarily mean that you actually have x µg in your sample. It is a useful beginner's exercise to determine the protein concentration of the same sample with different methods and different standard proteins, just to get a feel for the possible error.
For most applications, the colour yield cancels out as a constant factor, as long as you always use the same method and the same standard protein. You also need to identify both in your publications.
Check https://www.labome.com/method/Protein-Quantitation.html
Yes, we can estimate the accurate concentration of unknown protein by Bradford assay with a known standard.
The most correct I think is to purify the protein to homogeneity, freeze-dry the protein and measure its weight, and by knowing the mwt (kD) you can calculate its molarity or M
see
https://www.bioline.com/media/calculator/01_04.html
Importance of protein molarity calculator
Protein molarities are used in many contexts. Molarity tells us about how many proteins are present in a solution, where concentration just gives us the amount of mass of a protein in solution. But as mentioned before, the molecular weight of a protein can vary by orders of magnitude! This means that Molarity is a useful way to understand how much protein is in a solution without having knowledge of the molecular weight of every protein. In laboratory experiments this can mean having a physiologically relevant amount of a protein present in an in vitro experiment.
from
https://www.sepmag.eu/blog/protein-molarity-calculator
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