Hi, can anyone share a good refolding method for TGF beta3 expressed in E.coli as inclusion bodies? We have tried using a refolding screen test kit from Pierce, but we get protein aggregation/precipitation upon addition of solubilized protein samples to the buffers from the kit.
I would greatly appreciate any suggestions regarding this.
Check whether the protein becomes monomeric after the refolding by gel-electrophoresis in PAAG. In mixture after refolding. Does the molecular weight correspond to the predicted. If appropriate - refldding was successful. Replace the protein buffer. If the band in the desired area does not appear, change the conditions for refolding.
Laemmley electrophoresis, but use a denaturing agent without 2-mercaptoethanol, DTT, etc.
if possible dialize the mixture after renaturation against the acid buffer, for example, acetate, pH 4-5. When acidic pH values are suppressed, the process of formation of S-S bonds, which formed between protein molecules.
- Badges
- Science method