smhall wrote:If you are following some kind of published protocol regarding how ferritin (I am guessing you have a recombinant in E coli host) elutes from strong anion exchange on a Q column, then I could understand why you only increase the ionic strength to 0.15 M. As a general rule, I would always make the B buffer to have 1.0 M NaCl and run a steeper gradient at the end, usually as a cleaning procedure. In fact, if you do repetitive runs, I would probably not even regenerate with 0.5 M NaOH if I really did not have to do so.
Poster leekaming suggests checking if the proteins have the same relative mobility, possibly indicating they are identical. However, one of the proteins might be missing a few residues on either the N- or C-terminal end, and SDS-PAGE does not resolve that. Your best approach is to treat the material from the two peaks, digest them in trypsin, and analyze on C18 HPLC to look for the profiles, taking note of peaks present or absent at the different retention times. Further protein chemistry techniques might account for difference in affinity to the SAX column, including estimating thiols and disulfides of Cys and cystine, and post translational modifications (phosphates?)
smhall wrote:Poster leekaming suggests checking if the proteins have the same relative mobility, possibly indicating they are identical.
Users browsing this forum: Google [Bot] and 3 guests