Not observing a complex on a gel filtration run for a week complex
(micromolar) is not necessarily unexpected. It all depends on your
protein concentrations, the dissociation kinetics (remember, a gel
filtration run is not an equilibrium experiment - it dilutes and
separates your sample), etc.
I would make sure that the SPR results are believable and sensible: if
the affinity is really weak, I'd expect very fast kinetics - most weak
interactions come with faster association and dissociation. As a result,
in this affinity range, you can't fit kinetics data in SPR, and can only
do an Langmuir isotherm fit to your equilibrium binding response values.
Also, you should check your expected Rmax values (based on how much you
captured on the chip), and make sure that your responses don't exceed
the theoretical values. Otherwise, you are just observing non-specific
binding. Similarly, if your responses are way too low compared to the
expected, your protein on the chip might be mostly inactive - again, not
good.
If everything checks out, just mix your two proteins at the right
stoichiometric ratio, concentrate a lot, and go ahead with
crystallization (if that's what you want).
Engin
On 1/31/17 10:05 AM, sanjeev kumar wrote:
Dear all,
I am trying to stabilize a protein-protein complex. Our SPR study
indicates it is having micro molar dissociation constant. I tried to
purify both the molecule in complex form with size exclusion
chromatography (mixed both the protein in equal molar ratio and
incubated at 4 degree for 1 hour), I didnt observed formation of
complex as both the molecule eluted at their respected elution volume.
Please suggest me to get a better way to achieve the complex and if
anyone gives idea about what is the good cross-linker I can use.
Suggestions are highly appreciated.
Thanks
best
sanjeev kumar, PhD
Purdue University
West Lafayette
Indiana
