HIS TAG Protein Estimation - (Oct/28/2008 )
I am trying to purify my HIS TAG Protein. After purification by which biochemical method can we estimate the protein. I am using BCA method but its showing final concentration either in -value or very low. But i am seeing a good band on the gel. My standards are working good and the regreesion is also good.
Any help please.
usually protein estimating methods r not very accurate.U will see a band even if ur protein concentration is low
so how can we quantitate our protein. And it is showingn the concentration in negative values adn i am able to see a very good band.
as mentioned before, u can get low or negative value of protein concentration although u see a clear band of ur protein.
What u can do is using standards on the gel- meaning load different concentration of known standard (BSA) on gel and estimate
ur protein concentration compare to the standards (wide of the band on the gel).
Yep, amtash gave a good idea.
Also, I think you could try to remove imidazole from your sample and then measure the A280 and estimate your protein concentration.
Sandy, try removing Imidazole before BCA or try Imidazole from Merck chemicals, I recommend it!
you can also try comassie 250-G estimation.. bradford assay.