Hi, Sounds like your proten gradually aggrgates. Seems tat aggregation is exacerbated by disulphide crosslinking. There might also be a conformational change involved or perhaps one of your dimers is a 'true' dimer (for a given value of true) whilst the other one might be a disulphide- or metal- mediated. I would not mix the imac peaks, no. Some thoughts: attempt different resins for imac which allows you to play with imidazole concentrations for elution (some resins elute as low as 80-100 mM). Add edta right after elution. Add tcep instead of bme and see if things change. Have you already attempted gentle proteolysis? Do you have an activity assay?
Good luck, Artem On Mar 1, 2012 7:06 AM, "rashmi panigrahi" <rashmi.panigrah...@gmail.com> wrote: > Hi all, > 1) > I have a protein which gives two peaks on the 1ml Histrap column, Has > anyone seen this kind of behaviour and does this mean that there are two > populations of protein. They are partially seperated. > 2) > I tried to load the two peaks seperately on the superdex-75pg column. > They came out as roughly dimer but the difference in the peaks is 6mls > According to calculation with gel filtration standards > one was 1.8mer > and the other was 2.3 mer > Will there be problem if I mix the two peaks and load on the S-75 column?? > 3) > The protein is in 50mM HepespH7.3, 500mMKCl and 10% glycerol and imidazole > when it comes from the NiNTA column, > It is loaded on the superdex with same buffer but no imidazole. I get the > dimer peak. > If I concentrate and leave it, it start precipitating the next day even at > 2mg/ml. > I added bME (2mM) after the protein came out of superdex, and there was > no precipitation. > > Hence for the next prep, I did the superdex run with bME in the buffer, > there was a dimer peak and a small peak coming at the 125mls which is more > than 1CV (S-75 is a 120 ml column). Loaded this small peak on the gel and > it gave the same size band my protein. It could be my protein ... > > Hence I took the dimer from the above run concentrated and reloaded back > on the same column and there was a very tiny peak for dimer which was 10 > mAU for 0.5mls which is very less comapred to what I loaded. > > Does it mean that my protein is unfolded because of bME??? > > Any idea to stop precipitation would be helpful. > > with regards > Rashmi > > > >
