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HELP is needed for low expression using pEXP5-CT vector in BL21 (DE3) plyss


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#1 boginei

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Posted 19 February 2011 - 07:30 AM

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.

#2 priyaa Madhukaran

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Posted 22 February 2011 - 03:08 PM

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




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