A280 and ATP - Bradford or A280? (Feb/02/2006 )
i work with a 58kDa protein that forms a homohexamer and has autophosphorylation/dephosphorylation activity. during purification 1 mM ATP is used in all buffers for stability of the protein.
when measuring the protein concentration we routinely use BioRad's Bradford assay because ATP absorbs at 280nm. why can't we just blank with at 280nm with the ATP containing buffer and then measure the A280 of the protein sample? or is my kentucky-fried graduate student brain missing something simple?
hm, i don't know exact numbers, but a 1mM ATP solution should have a quite high absorbance at 280nm, which could mean that your instrument is not measuring correctly in that range (usually higher than 2 absorbance is not exact anymore with most instruments), even if you blank.
Kersten is right. The relative absorbance due to your protein vs the one due to ATP, would not be particulary significant at 280nm.
thank you for your replies!
the reason i assume i can blank it out is because 1 mM ATP is in all of the buffers used for FPLC purification and to "see" where the protien is, i just auto-zero when equilibrating a column. when the protein elutes it is a normal looking elution profile.
I would say that in FPLC, the protein is "concentrated " in the peak, and then diluted in buffer in the tube...
why don't you try.
put a known quantity of protein in ATP buffer and read it while blanking in ATP buffer.
what a nice experiment for today !
don't forget to tell us
if you just want to see, when your protein comes of the column, it doesn't mater, if the reading is exact. if you want to quantify, that's a different story, i would say.
furthermore, do you know the exact absorptioncoefficient of your protein at 280? may be that's also a reason why you use bradford