Protein expression troubleshooting - (Aug/26/2004 )

I expressed a mouse protein in E. coli -LB-kanamycin. It worked by SDS-PAGE and Western blot) but the expression was not very high. and I froze down my cells. Now, I have been trying to re-express my protein again but It is NOT working. I have included a positive control of another protein. everything seemed fine but I cannot see my protein again.
Something is masking the protein expression.
I even transformed the recombinant construct to a new expression host (E.coli DE3), and did everything just like last time, But No result.

Any hints!!

Hello
If you can not see your protein expression and getting expression for the positive results, this means that your operon is staying closed in the conditions where you are performing expression. My recommendation is to change the conditions of expression like temperature, induction time... If you can give more details about expression, we can make better thinkings, like your plasmid, temperature, amount of rare codon of the protein, IPTG concentration.
I hope you will express it. Good Luck
Mehmet

Thank you Mehmet for your input,

Finally, I got it back to work again. Basically, I went back and changed everything like (New LB medium, Kanamycin stock, IPTG stock, and I began induction at OD600= 0.8 at 37C for 5 hrs. I was able to see a thick band at my expected size compared to uninduced flask by CB staining.

I guess one or more of the above reagents were at fault.

Thank you again for your thoughts

Mbiologist 1