Protein expression with IPTG in pET3a system - (Jul/18/2002 )

I have tried the expression of a 20kD protein, cloned in pET3a in BL21 system. Bulk cultute is grown upto 0.5 OD at 600 nm and IPTG is added in 3 different conc. 0.1mM, 0.2mM and 0.3mM in 3 different sets and culture is collected after 1hr and 2hr induction. But I could not see the desired band in SDS-PAGE. Please give me suggestion.

-Jhimli Dasgupta-

QUOTE (Jhimli Dasgupta @ Jul 19 2002, 01:15 PM)

hi jhimli

u can induce with 1mM IPTG and should also reduce the temprature to 30 at the time of induction.
may it help you

Pranav

-HEV-

You can go up to 1mM of IPTG, and you can induce up to 4h... Now, when you say you can't see any of your protein, are you checking the total protein or only the soluble fraction?

-UniSPheryk-

I agree with others

you may also have a low expression, not always easy to see directely with coomassie blue. not all proteins can be exressed with high expression. depends on toxicity of the protein for the bacteria
did you check with WB?

-laurence-