Thursday, July 22, 2010


Sometimes, feeling hopeless and demotivated when things go too slow...

1. E. coli Rosetta-gami2(DE3) pET32-phaCcs
not stable, the expression level keep reducing after sometimes.
Revival of glycerol stock and verification ate most of my times.
Production is still OK. Some part in my mind is asking me to retransform the plasmid again.
However current priority is still crystallization(produce protein fast), and my target for crystallization is end of MAY!

2. Own stupid mistakes(thinking not diverse enough)
Purification in July
The purified PhaC protein seems not pure enough, so, I'm thinking to purify the elution again using the regenerated resin and I did.
However, the results of the SDS PAGE analysis is not good, because the PhaC protein cannot bind efficiently to the resin.

I thought is because of the Regenerated resins. Hence, I try new resin (mistake: I missed something very important here when troubleshooting).

For the 3rd purification, I used the elution from the 2nd purification. The result was the same. Binding of the protein is not efficient. Cannot figure out, think and think and think, and finally....I'm feeling stupid.

There are imidazole with concentration of 250mM in the elution that i used for 2nd and 3rd purification!

My large-scale protein production end here. no more protein. Now speeding for protein production again. I want xstal!

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