Any suggestion why the protein i'm tring to purify (E.coli expression) doesn't bond the resin? Same problems putting the 6XHis to N- and C-ter.
Hello Alessandra,
It seems the quality of your resin is not good. If the resin works with the positive control then its fine otherwise you need to recharge it. Also check your buffer composition for instance whether it has EDTA or other chemical which inhibits binding. Also verify by western that His tag is intact. If all these possibilities are ruled out then may be both the N terminus and the C terminus are in the interior of the protein and are not accessible for binding. In that case think alternative strategies to purify. In some cases adding ATP, specially for the replication proteins helps in binding.
Thank you Ram; actually i work with "fresh" resin and EDTA is never added in the purification steps. The clone was sequenced and the His tag was intact and in the right position of the protein.
Can you better explain the ATP addition? Do you mean adding it into the lysis and purification buffers? Which quantities?
Tnx
Hello Great! Yes about the ATP. I used to work with e.coli DnaB and it also did not bind with the resin. So I used to add ATP in the lysis buffer, 2mM and then it used to work to some extent. Had you checked other components of lysis buffer whether they are comparable with the resin. If all are fine then it tells you something about the structure of your protein, that is both the ends are in the interior of the protein and are not accessible for binding, this might also arise if the protein is multimer. If all these methods fails then add a bigger tag that is 10-15xHis or GST. If that also fails then try to purify by native methods, since your protein is tagged with his tag so you can easily follow.
good luck.
It is very difficult to ensure frame shift in clone through sequencing only. And due to frameshift you may not get His tag. Have you matched complete sequence of gene starting from promoter sequence of Vector? please perform westernblot, as suggested bY Dr Ram if you have antibody either against your protein or poyHis.
Thanks Gopal: actually the clone was expressed, so no problem with frameshift.
Hello Alessandra, I can see two different aspects affecting the experiment. The first one is fold of your protein causing that the His-tag is hidden in the structure and not exposed to the resin. I would try different composition of the lysis buffer- salt content, detergent, etc... The second aspect is that somehow the resin is affected and not able to bind your protein. So what kind of resin are you using. What metal is used to capture your protein. Are you using a reducing agent like beta-mercaptoethanol or DTT. These can reduce the metal in the resin, and it is no more capable of binding the target protein...
Hello Alessandra,
Make sure to include protease inhibitor in your lysis buffer like PMSF to avoid cleavage of the Tag which would explain why your protein is not binding to the column.
You could also try to re-design your construct to include a linker between your protein and the Tag to make it more accessible. You could try few residues, a Glycine/Serine linker or or a protease cleavage site (like TEV) if you want to remove the Tag after purification.
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