From SV-AUC, I got the Kd value close to micromolar range which shows rather a good affinity between two proteins. however, I am not able to purify complex for structural studies. What could be the reasons for this problem? I am sure for my SV-AUC data and have doubts on purification of complex, what can I modify? I use size exclusion chromatography, with low flow rate to get well seperated peak for the complex. I want to know the stichiometry of proteins in the complex. What possible techniques I can consider to do for knowing this?
Chemical crosslinking may be helpful to study the stoichiometry. The idea is to treat the complex with a water-soluble bifunctional amine crosslinker, such as BS3 (bis(sulfosuccinimidyl)suberate), using a range of crosslinker concentrations. The crosslinked sample is then run on SDS-PAGE. You are looking for a consistent mass over a range of relatively low crosslinker concentrations to indicate the actual complex size. (At high crosslinker concentrations, larger complexes may be formed that are not physiologically relevant.)
Other methods that can give you the size of the complex, if the equipment is available, are mass photometry and dynamic light scattering.
If the complex doesn't stay together during chromatography, maybe you can purify it using sucrose gradient centrifugation.
Changing the conditions may improve the stability of the complex. pH and salt concentration are two obvious factors to test.
Is the complex being dissociated during purification? What was the buffer recipe you used for SEC fractionation? Does it have destructive content or just in PBS condition?
Try native-PAGE as an alternative and calculate the MW of the complex to figure out the stoichiometry by taking into account the monomer MWs.
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