I have two proteins of interest - one is GFP-tagged and one is mCherry tagged. Each protein has several different forms (wild-type, mutant).
My hypothesis is that the wild-type (not the mutant) of the GFP- tagged protein will bind more readily to the mutant form of the mCherry-tagged protein.
I co-expressed the two together in HeLa cells and pulled down for GFP - however I see different thickness bands every time and no definitive result of whether it binds more or less.
As I am doing a transient expression, I presume the transfection efficiency and other factors could play a role in getting variable results.
From my understanding, co-IP is not the best method to quantify changes in the binding.
I was wondering if anyone could suggest a more robust way to quantify these changes.
Thank you!
I am guessing you are running your whole lysate too, and if you see similar expression of both your proteins, then you can attribute the thicker band to more robust binding.
Also I am a little unclear about your experiment? Can you give more details?
Have you considered using a flow cytometery approach using a flow cytometer which has a channel for detecting your GFP and mCherry signals?
So if you were to do this hypothetically do this- you would need the following controls.
(a) Nontreated- Hela cells
(b) Hela-cells with your proteins of interest (but not fluorescently tagged)
(c) WT Gfp pulled down Hela cells (not co-expressing any mcherry protein)
(d) WT Gfp pulled down Hela cells (co-expressing mcherry WT protein)
(e) WT Gfp pulled down Hela cells (co-expressing mcherry mutant protein)
(f) mutant Gfp pulled down Hela cells (co-expressing mcherry WT protein)
(g) mutant Gfp pulled down Hela cells (co-expressing mcherry mutant protein)
(a) and (b) are controls to be used to cancell noise Gfp and mcherry signals in the flow cytometer. If (a) and (b) have been set up correctly, with (c) you should be able to detect signals in" GFP+ mcherry-" quadrant of flow cyt. And if your hypothesis holds true, you can hopefully detect signals in "GFP+mcherry+" quadrant for (e), but not for any others (c), (d), (f) or (g).
Maybe this might work?
The problem with the experiment you are trying to do is that even if the mutant is binding with reduced efficiency, you will still observe binding in the absence of competition. The correct way to do this is a competition experiment where you probe binding of the labelled mutant in the presence of excess wt protein, and the reverse. I realize that this may not be possible with the proteins you are working with, but think of a different experimental design. Perhaps you can express each protein in different cells and probe binding upon mixing lysates together? That way you can play with ratios.
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