Hi Faisal,
When you say:
> On size exclusion chromatography a trimeric complex is obtained (something around 50 kDa). Now the question is whether the complex is 2X and one Y or 2Y one X ?
Are you simply estimating size based on elution volume? This can easily give an anomalous result if your complex is not perfectly globular. You might consider using something like MALLS to get an absolute mass. I just ask because perhaps this is just an XY dimer and the complex is a little elongated.
Can you get the two proteins to resolve in SDS-PAGE? You could plausibly resolve the stoichiometry by careful analysis of the band intensity.
A distinct approach, and an alternate to the already suggested ITC, is AUC followed by multiple signals. You can get approximate mass and relative stoichiometry with enough accuracy to distinguish these two models. It has worked well for me in several challenging cases. The method does not require tryptophans, but they may help, as would an introduced chromophore in one species.
Shae Padrick
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