I am trying to purify one protein in 2mL agarose-NTA-Ni2+ column at flow rate less than 1mL/min (equilibrated in 100mM phosphate buffer pH 7.4 with 1M NaCl).
Multiple peaks can be observed in flow-through. SDS-PAGE indicates first peaks contains higher and later peaks contain lesser MW proteins respectively. Any explanation?
I will assume that you are trying to purify a protein with a His-tag. What might be hapening is that your protein is being cleaved in your crude, leaving you with smaller fragments of your protein attached to the His-tag. According to your SDS-PAGE results, it would appear as if your protein corresponds (hopefully) with the first peak to elute, and the other peaks are partially digested fragments.
In order to avoid this problem, I would recommend that all prior treatments of your crude as well as the purification are performed at 4ºC to minimize the protease activity that is probably present in your original mixture. Another solution would be to add protease inhibitors, commercially available from Sigma Aldrich or Bio-Rad, for example.
It depends on what peaks are coming in your flow-through. You did say flow-through and not elution, right? If the peaks "are" your protein (via western or such), then you have a proteolysis problem. If you're just seeing unwanted proteins coming out in peaks, it may be that you have a sizing effect due to the beads: unbound proteins that are large come out in the void volume and smaller ones are slightly retained on the matrix, like Sephadex would do. Not a problem.
Hi Ricardo,
its an IMAC column, so question of sizing effect is not that much. Here my question is not exactly "my protein", anyway it is coming in the elution. I am astonished by seeing two completely separate peaks in flow through, although amount of protein in the load is also less. I couldn't reason it, thats why I put it in public domain, if somebody has observed it earier.
Right. I'm aware that it's an affinity column, but that does not eliminate the potential for sizing behavior in the beads. Either way, it's not a problem, apparently; just a curious phenomenon.
I also Agree with Sergio. This type of problem also with me,but this problem is troubleshoot by adding Protease inhibitor in crude as well as whole experiments are performed at 4 degree.
The size exclusion properties of the beads do no disappear just because a ligand is attached. But most likely you have a quite short column, so the effect will be small. Still if you have large aggregates and perhaps DNA present that is completely excluded from the beads you may see the flow-through split into two peaks because of size. But you will see it only if your sample volume is small relative to the column size. For IMAC the sample volumes are usually large compared to the column volume. Another effect you can see sometimes is weakly bound proteins being displaced by the strongly binding his-tag. That could manifest as multiple peaks in the flow-through.
Agree with Ricardo and Bo, it's probably the size exclusion effect. I've seen it few times, although rather that the change in conductivity appeared later than flow-through proteins.
might be due to the rapide flow rate or presence micro cracks or fissures in your coloum or protease processing phenomena which can be avoided by adding Protease inhibitors
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