I have cloned a gene in pET28a vector, and induced it for 5 hrs with 1mM IPTG. The cells were centrifuged from 1 ml culture (Uninduced and induced both), and the pellet was suspended in 200 microlitres 2X SDS loading dye and kept at -20 overnight for further use.
The next day, run on 12% SDS PAGE, on staining induced band was visible.
To have a better image, the same protein was run again on 12% SDA PAGE, the induced band disappeared.
pls suggest, how to stabilize induced protein.
Hi, it is hard to nail down the reasons because you did not purify your protein. When you resuspend with 2X SDS solution, this should denature all the proteins in the solution (such as protease enzymes) so that no enzymatic degradation will occur. You could not mix your sample well when you repeated the run. Try to boil your sample for 5 minutes when you add the SDS, and keep the rest at -20 for further use. Also, try to purify your protein and study its stability at different temperatures and buffers.
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