hi,
i am trying to purify an 8kda protein.PI 6.95, HIS sumo tagged.
after adding sumo protease to my elution sample plus 2mM DTT .DAILYSIS BUFFER being 20mM NaH2PO4 500mM NaCL..when i again bind my sample to nickel column there comes nothing in flow through. when i add 1omM or 20mM immidazole to elute both my protein and sumoprotease co elute. sometimes only sumoprotease elute. can anyone suggest me anything?
Could you please be more specific. What are the conditions when both proteins co-elute? What is different when only the protease elutes? Are there any other superficial histidyl residues in your protein? What about the protease?
I think you should purify your fused protein first; and after you know that your whole purification process was okay by testing the purity of your protein using SDS-PAGE, you can add the sumo protease to remove the tag. In other words, a better way to remove the tag is after purification not during it.
Good luck
@Aamal I have purified my fused protein and got the band on SDS PAGE .after that i added excess of sumo protease and 2mM DTT( to increase the activity of sumo protease enzyme) to my elution sample and at the same time i put the sample into dialysis bag and dialysed it in dialysis buffer (20mM NaH2PO4 + 500 mM NaCL pH 8). i again ran the sample on SDS PAGE to see whether sumo protease cleaved the tag from my protein or not. I observed that the his sumo tag was removed from my protein.I then again loaded the sample to the nickel column to collect my protein .it should be in flow through but i didnt observed anything in flowthrough .but when i added 20mM immidazole to the column.both sumoprotease and my protein coeluted.how can i separate them because AKATA system can alo not separate them
after marker, the first lane is my sample after dialysis i.e sumoprotease and my 8kda protein, 2nd lane is flowthrough,third lane is 10mM immidazole eluate, 4rth lane is 20mM immidazole eluate ,5th lane is eluate with elution buffer (20mM NaH2 Po4+ 500 mM NaCL 500mM IMMIDAZOLE and last lane is eluate with edta
@Omar .I USED pET28a vector with sumo tag. we use our own sumo protease prepared in our lab.the problem is that why cannot i get my protein in flow through once it has been cleaved by sumo protease.
Run a slow imidazole gradient from 10 to 500 mM and you should be able to separate your protein. Answer my previous questions please and they will give insight in what is happening. Also try cobalt and zinc ions to see if separation is favored.
Anam Nayab I am facing the same issue as you had. Did you manage to purify your protein using nickel?
Maria Beatriz Takahashi I changed the vector.you can also try using different vectors.
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