I am trying to use FRAP to measure the binding kinetics of two proteins. A is an immobile protein, which could be bound with B (mobile protein and with fluorescent tag). I photobleach one region with A and B bound, then wait for the recovery and measure the time course of FRAP. After repeating it under different concentration of B, I try to extract the on/off rate of B binding A by using the following equation:

K(FRAP)=Kon*[B]+Koff

My question is, can I do experiments like what I describe? Any criticism and concerns about the experiment design? thanks!!!

Sounds very interesting,
I would first show that the tags are not changing the fanctionality of the proteins. One by one and then together.
You can try other means if this is not working.
Have you done some IP studies to show the binding of the two proteins?
Pulling down a and looking at b and vise verse?
Guy

how do you differentiate between Free Fluo-tagged B protein diffusing back into your bleached region and Fluo Tagged B protein that diffuses in and binds A?

Are you doing just FRAP or FRET/FRAP or TIR/FRAP?

Look up Daniel Axelrod's work (University of Michigan, Ann Arbor).

Sund SE, Axelrod D.
Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching.
Biophys J. 2000 Sep;79(3):1655-69.
PMID: 10969025


Mc Kiernan AE, MacDonald RI, MacDonald RC, Axelrod D.
Cytoskeletal protein binding kinetics at planar phospholipid membranes.
Biophys J. 1997 Oct;73(4):1987-98.
PMID: 9336194

Stout AL, Axelrod D.
Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.
Biophys J. 1994 Sep;67(3):1324-34.
PMID: 7811947

Yes, the tag would not change the function of the protein, that is the basic but important control experiments to do.

That is an excellent question, i do not think I could differentiate between bleached-tagged B protein and nonbleached -tagged B protein. But I tried to avoid this problem by constant superfusing the cells with fresh tagged B protein. Sorry, I forget to tell you that my cell has been permeabilized, and I could introduce tagged B protein by superfusing the cells. Another important assumption here is, the perfusion is fast enough to avoid the rebinding problem. Please let me know what do you think of this modification? Thanks a lot.