Dear all,
I have some questions about the elution process of GFP-trap agarose. What's the best way to elute the protein and keep the enzyme activity at the same time since the eluted protein need to be quantified and done the further enzyme activity assay?
From what I've read, this resin is intended for elution under denaturing conditions with SDS and low pH. The antibody's affinity is very high, with a dissociation constant of 1 pM. You might be able to compete the GFP conjugate off the resin under non-denaturing conditions by incubating for a long time with purified GFP. If that works, extra added GFP will be mixed in with the GFP conjugate.
Adam B Shapiro Dear Adam, thank you for your constructive suggestion. since my target protein contains gfp, I am not sure about the efficiency and also if extra GFP will be mixed with my fused GFP-protein, will this influence the following enzyme activity assay. Because the buffer and other chemical may influence the reaction which remain unsure at all for me. Do you have experience of gfp compete and any publications you can suggest?
Given that the binding between GFP and nanobodies is extremely tight, you can try to perform on-column digestion provided you have introduced a TEV/HRV 3C cutting site between GFP and your POI. Depending on the molecular weight of your POI and the protease, you can remove the latter by gel filtration. Alternatively, nickel or cobalt column is an option if your protease is His-tagged. Or proceed to your next step if the protease does not interfere with your assay. Just remember to double check if your POI does not contain sequences recognized by the protease.
GFP does not have any enzymatic activity, as far as I know.
It could interfere in an assay that relies on measuring fluorescence, if the excitation and emission wavelengths are similar to those of GFP. The result would be a constant background fluorescence from the excess GFP.
A protein concentration measurement would detect any excess free GFP, which would reduce the specific activity of your fusion protein.
I like the suggestion of Wojciech Woj Bialek to separate the protein of interest from the tightly bound GFP on the resin by introducing a specific proteolytic cleavage site, such as TEV.
Here comes the most straight forward suggestion: For the purpose of enzyme assays, there is no need to elute the GFP fusion protein. Just do an assay with the enzyme still on beads. I have sone this several times. However, there is no good way to quantify the protein amount. What you can do: After the assay, elute under denaturing conditions and analyse the amount of enzyme by running samples on a Western Blot.
Best wishes Jürgen
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