his purification

shifali chatrath via methods%40net.bio.net (by shifalich from rediffmail.com)
Wed Jun 9 06:22:39 EST 2010


I always get a monomer and a dimer band (sometimes multimer bands also) of my protein in reducing SDS-PAGE even if I purify my protein under denaturing conditions (6M gnHCL). but monomer mass if I purify protein on reverse phase HPLC followed by ESI-MS.So, I doubt if GnHCl or urea will help him for sure. I mean Siva can always try but I wouldn't be surprised if it doesn't work.ShifaliOn Wed, 09 Jun 2010 03:41:36 +0530 wrote>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" 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 from net.bio.net>http://www.bio.net/biomail/listinfo/methods>


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