I recently purified two GST tag fragments that are below 100 amino acids and did pull down assays. However, neither of them interacted with the other purified protein. We know that the two fragments should interact with the other protein since we've done a truncation of the fragment from the FL protein and it abolished the binding. I wonder if the fragments might form soluble aggregate and it prevents the interaction with the other protein. Does anyone have any advice on this problem?
Hi Lan,
I agree with Alim. Your 100 amino acids might not be properly folded depending on how you did your truncation in the first place. Do you have any structural insight about the proteins or the 100 amino acid fragments? You could try to do CCD Spectroscopy to check if your 100 kDa fragment shows any content of helices or beta sheets.
Thanks everyone. Both of the proteins are purified proteins. I'll do a SEC later with this GST tag protein. Since I've purified a larger truncation before and it will always forms something around 6-10 molecules or more, so I assume that this smaller fragment will also form some aggregation. From the structure analysis, we know that this fragment is composed of a lot of beta sheets.
I agree with the previous suggestions. Three main things could be occurring, perhaps more than one of these at the same time:
1. The peptides binding site was lost by cleavage
2. The peptides are self-aggregating
3. The peptides are not correctly folded and the binding site is hidden
Since you believe there is aggregation going on, I would suggest adding a small amount of a non-ionic detergent to prevent aggregation. After aggregation you would need a stronger chaotropic agent to disrupt the aggregates. Good luck.
it just came to my mind. If your peptides have a reactive cysteine (and this cysteine is important for binding) oxidation of the cysteine or dimerisation over the cysteine can occur. This is however a very special case.
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