~Progress~

Previously, I informed that induction at 28°C give more soluble form compared with other induction temperature. While M15-pQEC1/pGro7 give better result than M15-pQEC1.

Thus, my attempt is to grow the cell at 28°C until it reach OD600 reach 0.5 -0.6
(midexponential phase),and also the induction was also at the same temperature.
*to promote slow grow of cell so that the cell was not force to express the protein, and allow the protein to fold properly in order to get higher amount of protein.

Results:
1)I have done solubility test and also purification. All the elution give approximately a thick band at around 60 kDa.Its indicate that my protein is quite a lot.

2)Since there's a lot of contaminants, I've decided to do gel filtration (size -exclusion chromatography)to get purity of protein.

Problem:

~There's no peak of elution appear after filtration was done.I redo the filtration but I got the same results. Where is my protein????

Troubleshoots:

~ May be my protein precipitate in low concentration of salt. This is because, I notice that [NaCl] in the buffer use for filtration was lower compared to the one in the buffer used for Affinity Chromtography.It's my mistake..
*Redo gel filtration with the same and/or higher [NaCl] in the equilibration/wash buffer

~ My protein was degrade since it's a week olds.
* Rerun the SDS-PAGE to check the stability of the protein. I've noticed that my protein which supposed to have MW of 60 kDa was now at ~30-40 kDa. I think that's why I've got negative result for gel filtration.


-----I'll culture, expressed, and purify the protein straight away...Hopefully I'll success in my work-----