It is not surprising that your bradford and BCA assays don't agree if you have no aromatic amino acids in your protein. Bradford dye binds to hydrophobic residues, mainly aromatics, so I would guess your bradford is consistantly giving lower measurements than the BCA assay. I also wouldn't be surprised if the results of your Bradford vary significantly between replicates. The BCA assay reagent interacts with the backbone amides, not with any sidechains, so I would tend to believe that measurement more than anything else you have done.
I work with a protein that has very few hydrophobics (only one aromatic - a Phe) and I have found that Bradfords are unreliable, but the BCA assay tends to be consistent. Mike ----- Original Message ----- From: "Arpit Mishra" <ar...@igib.in> To: CCP4BB@JISCMAIL.AC.UK Sent: Saturday, April 9, 2011 2:52:21 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] how to quantitate protein which dont have ne aromatic residue hello everybody i am working on the protien which dont have any aromatic residue i do fplc other purification using 220 absorption, but i want to quantitate protein precisely i have tried using BCA nd bradford but both methods quantification is not matching,,so any one is having sum idea how to quantitate it precisely thanks in advance for your valuable suggestion.. Arpit Mishra -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
