Hello
Despite the many treatments that I gave, I didn't even get missfold peotein(Dimer).
I see a lot of monomer on the electrophoresis gel (sometimes as a single band and sometimes as two bands), but despite the different refolding conditions, it still does not become a dimer.
How that is possible, they have 5 cys and must do dimer.
Can Romiplostim monomers become irreversible oxide during the production process( fermentation and lysis) so, cannot be refolded anymore?
thank you
The electrophoresis method you are using is a form of native PAGE that is known to work for your protein? If you are using SDS-PAGE, you can only (with few exceptions) see monomer, because the conditions are denaturing. The paper linked below suggests that the dimers are covalently linked by disulfide bonds under oxidizing conditions. If this is correct, you can see the dimer even on SDS-PAGE, but you have to omit the reducing agents (typically dithiothreitol or 2-mercaptoethanol) that most people routinely add to the sample loading buffer.
Article Expression, purification and biological activity assessment ...
Which method do you use to check for dimer formation, other than PAGE?
Dear Leonard
Unfortunately, the method written in this paper has not got more detailed; it was extracted from the Amgen patent. However, it needs to be optimized by using a time course
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