I think it's worth a try.

If the protein does not get trapped in the column (and then elutes together with the protein you use for fishing, when you're stripping the column), there is a high chance that it elutes delayed, of course depending on the buffer conditions.

Alternatively, you might try to covalently immobilize the bait protein, e.g., on NHS activated sepharose, which should give you more choices of conditions for binding and elution.

Hi Rebecca,

I agree with Wolfgana that it is worth trying. Just remember to include proper controls to exclude non-specific binding to Ni-beads.

In case your interaction of interest is weak/transient to be detected with IP, you might consider alternatives like BioID proximity labeling for example.

Good luck!

If you use cell lysate, the result cannot tell whether it is a direct or indirect interaction.

I think you would need a high expression level of your interesting protein to make it work.

Thanks for the responses! I was thinking I would try passing the lysate through just the Ni resin first and then proteins with affinity for the resin itself will be taken out (there are quite a few His-rich proteins in trypanosomes), then I pass the remaining lysate through Ni resin+ immobilized enzyme (fishing bait) and analyse anything which elutes from the second round (compared with a control). Hopefully something might be revealed as a preliminary test, if as Ahmed Soliman says the interactions are not too weak. And if nothing is revealed then I will try these great alternative suggestions

If you are interested in an emzyme-substrate interaction, such an interaction is usually transient so, you might consider cross-linking proteins in your lysate before IP. Also using a mutant enzyme that can entrap/keep the substrate would be ideal (if practically available). Good luck!

A Pull-down experiment is an effective in vitro experimental technique to verify the interaction between proteins. The basic principle is to affinity-immobilize the target protein on the matrix and act as a "bait protein". When the cell extract or other solutions containing the target protein pass through the column, the target protein that interacts with the target protein can bind to the matrix, and other non-interacting impurity proteins flow out with the solution. The adsorbed interacting proteins can be eluted by eluent or elution conditions, and detected by Western Blot or Mass Spectrometry. The method is simple, easy to operate, does not need to use dangerous isotopes and other substances, and has been widely used in protein interaction analysis.