Dear Siva. does your protein form multimers (do you see one or a few sharp bands/ regular pattern in a non reducing gel)? Or it just precipitated / aggregated on the metal column or during dialysis.due to unfavorable conditions You might try to change pH or ionic strength, type of salt (maybe KCl or MgCl2 does a better job), elute with EDTA (you'll need to recharge your column with the appropriate metal salt afterwards), add some (say up to 2 or 4M) urea or GuSCN or GuHCl or iodide. Also adding some mild detergent (eg 0.1...0.5%Triton X 100, Tween 20) or reducing agent (DTT, BME) might help, depending on the nature (cellular location?) of your protein. Is it sensitive to oxidation?
Actually, when performing size exclusion chromatography, you may skip the dialysis step as you will have a buffer exchange at the same time when you equilibrate your gel filtration column with the target buffer. Depending on the sample size, a PD10 column (2.5ml loading volume) might do this job, if you are afraid to spoil your big Superdex column. Good luck! Wo On Jun 8, 9:12 pm, "UTHANDI,SIVAKUMAR" <us...@ufl.edu> wrote: > Hi All > I am trying to purify His-tagged protein from a halophilic (2M > NaCl)archaea which is around 30kDa. I was succesful in purifying > thro nickel column. When I run the His purified protein sample > (after dialysis in Tris buffer containing 2M NaCl, dilaysis also > helps to remove excess immidazol from the protein) on to the > gelfiltration coulumn(superdex 200), it was eluting at a molecular > weight of ~460kDa. I dont know whether the protein is getting > complexed or aggregated? But shows single band on SDS Page. > Can any one has the similar problem? Any help please? > Thanks, Siva > -- > UTHANDI,SIVAKUMAR _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
