IPTG induction doesn't work:(IPTG pet28a expression
Posted 21 August 2012 - 06:34 AM
I'm trying to express 24kDa protein in Bal22 using pet28a expression vector. I've checked my construct, everything is ok as it supposed to be. However I have a problem: IPTG induction doesn't work. I'm growing the cells for 2h than I'm adding IPTG at different concentrations (I've tried 1-4mM) and then continue growing the bacteria for additional 2-4 hours. I did'n get the bold band at any IPTG concentration or any incubation time.. Should I try other IPTG concentrations or maybe the incubation time following IPTG addition should be extanded?
Will be very greatful for any advise
Thanks in advance
Posted 21 August 2012 - 07:13 AM
2. Do you add freshly prepared IPTG? Maybe your IPTG stock is old.
3. Induction time (and temperature) greatly varies from one protein to another. Try at least 4-8 hours at 30C ...take an aliquot every hour of induction. Alternatively you could try O/N induction at 16C
Posted 21 August 2012 - 12:58 PM
Are you using a DE3 bacterial strain for expression? pET28 doesn't work without the DE3 properties.
Not all the proteins are expressed with thick bands: what I am usually doing is a pull down using 20-50 uL of Ni-sepharose beads of the cell lysate of 20 mL culture resuspended in 2 mL buffer to be sure that my protein is expressed then WB anti His tag. Some proteins need another media or other temperatures. And less IPTG (4 mM is a bit too much). I usually do 0.1, 0.5 and 1 mM IPTG. temperatures 18, 22, 25, 30 and 37 oC 3h after induction and overnight. I test usually LB, TB, ZYM5052 and MD5052 (autoinduction media from Studier; google these terms)
Moreover: not all proteins are happy with a strong promoter such as T7. You could try milder ones such as BAD or TAC... or anything less strong. People usually try 3 constructs for 3 promoters X 3 different tags.
I recommend you read:
http://www.currentpr...fId-ps0524.html (for me this was mind blowing when I started my PhD)