Protein quantification with BSA? - (Nov/14/2007 )
To much people quantify its protein samples with a BSA standard curve.
But, this way to determine the concentration is the same as reading the sample OD and then apply the BSA extinction coefficient (which would be faster), isn't it?
-celvas-
QUOTE (celvas @ Nov 14 2007, 04:05 AM)
To much people quantify its protein samples with a BSA standard curve.
But, this way to determine the concentration is the same as reading the sample OD and then apply the BSA extinction coefficient (which would be faster), isn't it?
But, this way to determine the concentration is the same as reading the sample OD and then apply the BSA extinction coefficient (which would be faster), isn't it?
I don't know... what solution is your BSA extinction coefficient determined in? is it the same as your samples, is the background equivalent... Does DNA or RNA or something else in the sample absorb at a similar wavelength and therefore obfuscate your results...???
Anyway I kinda think all protein quantitation methods are a little misleading, (I mean all proteins in a cell will not interact with a quantitation reagent in the same relative ratio/way as BSA) so maybe your suggestion is a valid one, regardless I think it is fair to presume that no protein quantitation method is absolute and just be satisfied with relative concentrations so that all your samples are loaded at the same amount even if you really don't know what that amount is...
Oh well, my two cents... Hope it is helpful to you....
-beccaf22-
QUOTE (celvas @ Nov 14 2007, 08:05 PM)
To much people quantify its protein samples with a BSA standard curve.
But, this way to determine the concentration is the same as reading the sample OD and then apply the BSA extinction coefficient (which would be faster), isn't it?
But, this way to determine the concentration is the same as reading the sample OD and then apply the BSA extinction coefficient (which would be faster), isn't it?
Any spectrophotometric system that compares the sample to BSA (or any other standard for that matter) is totally dependent on the similarity between the two proteins. If BSA has a significantly different epsilon to your protein, the calculated values will be significantly different as well. Have you considered Bradford?
-swanny-