Basically, I like researcher's life, is always full of surprises and disappointments. Of course, very tiring and a very hard efforts are required. So far, There are 4 big stages of my project :
Cloning, Expression, Purification and the crystallization.
Cloning
Construct the desired DNA sequence into a suitable expression vector, and transform into a host, mostly E. coli.
In my case, I'm using pET32 expression vector from Novagen.
Transform into E. coli strain Rosetta-gami 2.
This stage done!
However, I'm trying another expression vector, pCold expression vector, and transform into classic of expression host, E. coli strain BL21 pLySs.
On the way!
Expression
To stimulate the bacterial host in order to over express (translate the gene seq on vector into protein) desired protein.
Optimizations were done, mainly on temperature. 15C, 25C, 30C, 37C. IPTG as inducer with concentration of 0.4mM.
Run SDS PAGE to determine the proteins.
Purification
Using TALON resin, going to try gel filtration soon. maybe can play ion exchange le? yohoo!
Others
6X His Protein Staining, Western Blotting, In vitro enzyme assay
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