We are working on a family of proteins that forms homo and heterodimers. We have GFP labelled one of the members and would like to test this against an unlabelled other member of the same protein family. The GFP labelled protein will initially form a homodimer with both proteins GFP labelled. Titration with a different, unlabelled, member of the family will replace one of the GFP labelled proteins in the homodimer leading to formation of a heterodimer. Can we study this using MST? Can we obtain Kds etc?
Also, we have performed MST experiments with a GFP labelled protein and obtain variations in fluorescence that appear to depend on the ligand concentration. We would like to verify this via an SD test. The SD test suggested in the nanotemper manual is not suitable for GFP labelled proteins. Is there a good SD test available for GFP labelled proteins?
Hi Thomas
You should be able to study the heterodimer formation, because your concentration of the labelled protein should be very low compering with the non- labelled protein In the experiment. We have tried to study the formation of an homodimer using the same protein labelled and non-labelled and we got good results so i think with the heterodimer should also work, if the Kd is not to high comparing to the one of the homodimer.
You can not use the SD test for GFP because what you do is denaturated the protein and then you will loss the fluorescence. But I suggest you that you try to add some additive like tween or glycerol, or use premium capillaries to be sure that difference of initial fluorescence is not due to stickiness of the protein.
You can always use chemical labelled instead of GFP. It is an easy and fast reaction and in my case has work a bit better than GFP.
I hope this helps a bit, good luck!
/Maria-Jose
Thanks Maria, great idea with the chemical labeling, we considered this and may have to go ahead with it as our current experiments suggest potential ligand induced changes b(i.e. the fluorescence changes with increasing ligand concentration) but we need to verify that it is not due to other factors using an SD test
Thomas
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