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HELP is needed for low expression using pEXP5-CT vector in BL21 (DE3) plyss - low expression after time course study with 1 mM IPTG (Feb/19/2011 )

I need help in protein expression. I had tried expressing my protein with 1 mM IPTG in BL21 (DE3)plyss cells. However I can only see very thin band in western blot. I do not know whether my protein is toxic. I had even tried expressing in top 10 cells. But the expected band was doubled up in size in western blot analysis. I really do not know what is wrong with my protein.

-boginei-

boginei on Sat Feb 19 15:30:07 2011 said:


I need help in protein expression. I had tried expressing my protein with 1 mM IPTG in BL21 (DE3)plyss cells. However I can only see very thin band in western blot. I do not know whether my protein is toxic. I had even tried expressing in top 10 cells. But the expected band was doubled up in size in western blot analysis. I really do not know what is wrong with my protein.


Hi,
I am working with 18KDa protein.
Generally i use 0.500mM IPTG to induce my protein
ie:If my stock is 1M IPTG i add 500ul of IPTG to 1Litre culture.
Calculation:
1L culture*0.500mM IPTG concentration/1M IPTG stock=0.500 ul

-priyaa Madhukaran-