Protein concentration and extinction coefficient - (Jan/13/2014 )
I have a question regarding estimation of protein concentration for a recombinant protein. I am using Bio-Rad DC reagent, which measures absorbance at 595 nm. To obtain protein concentration, I use a standard curve generated using BSA protein standard. Typically, I dilute my protein 2000 fold to obtain the concentration within the range. Here's my question:
If my protein's extinction coefficient is 22000 and for the estimation of its concentration, I have used BSA as a standard (extinction coefficient = 44000), should I double the value obtained for concentration in mg /ml, for accurate estimation?
Thanks in advance!
with lowry-type protein assays, you're reading a common colored reaction product, not the protein itself.
unless you empirically determine a different response of the reagent to your protein then using bsa is perfectly acceptible, with no correction factor for extinction coefficient necessary.
That makes sense. Thanks mdfenko!