During my experiment, I remove a 7Kda protein from affinity column after its cleavage using prescission protease in a buffer containing NaCl ,Tris and DTT. After running the removed protein on SDS-PAGE gel, two protein bands are detectable, one narrow band belong to GST and the other sharp band belongs to 7Kda protein. But after injection to cation exchange chromatography (elution buffer NaCl 1M, Tris 50mM), two peaks are observable (attached file), one shows my protein but what about the first one? Can anyone please tell me what it belongs to?
Dear Hakimeh
You wtrote: "two protein bands are detectable, one narrow band belong to GST! and the other sharp band belongs to 7Kda proteinafter injection to cation exchange chromatography (elution buffer NaCl 1M, Tris 50mM), two peaks are observable (attached file), one shows my protein but what about the first one."
Probebly i miss something, but why do yuo think that the first one could not be GST? If i'm right GST as acidic isoelettric point so is it possible that it not bind in the cationic exchange.
because I concentrate the first peak and nothing was detectable on the gel, no GST was observed. and I am not sure if such a narrow protein band can give such high absorbance unit, I have no experience in that case
Hi Hakimeh, What I could make out from your post is that; you expressed your protein of interest with a GST-tag, later you tried to remove it by using some specific protease that cuts at the junction between your protein of interest and the GST-tag.
If so, is the digestion complete (I mean do you see only two bands ( bands corrosponding to GST and protein of your interest) or a third band corresponding to GST-fusion is also visible)?
If the answer is in negative then it looks plausible that the second peak is of GST-fusion (uncleaved fraction of your protein).
If the answer is positive, did you check the charge on GST at the pH of your buffer? If it has positive charge there are chances that it would also stick to the resin and elute later in increasing ionic strength.
Third case could be the oligomeric status of your protein, it looks like you did not add DTT in the buffers used in ion-exchange chromatography, in that case you might get some a small fraction of your protein as oligomers which will have slightly different properties from the monomeric state and might get eluted at different ionic strengths.
I hope what I wrote here does make some sense to you! :)
thank you both very much.
you are right, GST is acidic then will remove in flow through as an unbound protein. but please have a look at my gel, can I expect that peak for GST from this sample? I have loaded 1mg of protein to chromatography
Hi Hakimeh,
The first peak is a very sharp one. It is the unbound fraction , i.e., protein molecules that failed to bind to the column. So, what caused them to do so? It is probably their conformation which have shielded their surface charge. Now, this conformational transition can be as a result of insolubles or microaggregates (soluble protein aggregates-remember you are loading high concentration of protein to the column). But why there is no band on SDS-PAGE even after concentrationg peak 1? It may be loss of protein during conc.
Anyway, It is a nice gel.
Best of Luck
What are the elution conditions? Isocratic or gradient/multistep gradient, flow rate, column size, etc.?
Are you sure that the first pic does not belong to the eluent (salts)? Have you done a blank before?
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