My protein after gel filtration has a tendency to oligomerize into numerous soluble ologomers. What will aid in shifting this equilibria of protein towards any single homomeric state, so that I can proceed with its crystallization? It is a transcription factor . Even concentration dependant oligomerization shifts have been observed. Also, changing buffer pH did not affecr the oligomerization state in solvent. what do you think might work for obtaining one stable state after purification?
In the absence of detergents and chaotropes, non-detergent sulfobetaines (NDSB) could help prevent hydrophobic aggregation and will improve the yield of your active proteins. I use it at 1 mol.L-1... for charge-dependent multimerizations the addition of salts such as NaCl is recommended. In the case of multimerizations caused by disulfide bond formations, reducing agents such as DTT could be used.
You need to determine if the polymerization is hydrophobic or due to disulfide formation.
You need to identify optimum solution conditions for enhancing the stabilty of your target proteins.
You need to identify optimum solution conditions for enhancing the stabilty of your target proteins.
I would suggest you to run a simple SDS-PAGE in order to experience different solutions and to determine the best dissociating agent for your protein prior to your chromatography separation
following suggestions:
1. add DTT/bME during SEC and check the aggregation status later, if aggregation is sue to disulfide, it will resolve.
2. Increase the amount of salt in the SEC buffer, increasing salt concentration reduces aggregation very often.
3. If you are using NaCl, try Na2SO4, that is a more stabilising salt according to Hofmeister series.
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