Hello all,
I am relatively new to the field of Protein purification and FPLC. So, forgive my naive questions.
I want to study if protein A and protein B interacts by SEC. However, the protein A and protein B has different physiological properties ( for example one is aggregation prone, other is not) and Buffer conditions (for example protein A has: HEPES buffer, 150 mM Nacl, EDTA) other has (Tris-buffer, 50 mM KCl and b-ME, glycerol).
How can I optimise this and come with a physiological solution for both proteins to interact?
Any help would be appreciated.
You can run them independently through FPLC note down the elution profile - ( If PI and stability allow then you can use the same buffer conditions). Then mix them together, incubate them and run that sample in a proper cut off SEC column. If they want to stay together then you will see a new peak early in elution profile which will correspond to the molecular weight of a+b together. We don't have any more details of your proteins so it is difficult to comment more regarding this. Well, if aggregates are very large they will not enter the column or if proteins are interacting transiently then they will not stay together.
First of all, look through the literature to find out whether there is any reports of A+B interacting and what buffer was used.If such information is available then start with the reported condition and alter if necessary.
If such information is unavailable then you can do a buffer screening using a 96-well plate to find out which buffer is suitable for your proteins using a temperature gradient in a PCR machine.. This method requires little protein... After establishing this then choose the appropriate buffer type and mix protein A and B and incubate accordingly (say 30-60 minutes on ICE) followed by centrifugation at high speed (say 17 000g for 10min and hopefully you see no precitation) then run this onto the right column (required separation range in kDa).. Also, run protein A and B in isolation as controls and overlay all three runs to campare - in theory your run containing protein A+B should elute earlier as it might appear bigger in size (bigger hydrodynamic radius) relative to each individual protein - in theory!
Lastly, perhaps mixing protein A to B will cause stability thus you might not have to worry about much.. Some proteins are unstable untill a binding partner is present..
All the best!
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