My protein show correct size but very light on SDS-PAGE.
In addition, Other size band is more bulky than my protein.
I'm express in BL21(DE3) at 25'C for 18hr after added 0.5mM IPTG because prevent inclusion bodies formation.
Can anyone suggestion???
Hi there,
Low yield ?
Standardize expression steps in Mini Induction (that is 5ml culture) :
1.choice of expression E.coli factory ;
Try with all the available expression strains of e.coli :
RIL, Codon plus, Rosetta, PLysS (note antibiotic resistant carried by indigenous plasmid gennerally chloramphenicol)
2. Standarize IPTG concentration :
Some proteins yield is high at low IPTG and some dose dependent.
3. Temperature :
Post Induction temperature influence the yield. If your proteins struggling to fold at uncomfortable temperature. Try 16 degree Celsius. And also send some helping hands of chaperone/ heat shock protein . So before giving induction subject your culture in shaker to 10 % ethanol or 42 degree for 45 minutes.( these conditions send wake up call for chaperones to induced to take of protein folding).
4.Play with all these conditions and run SD-PAGE to check the mini induction profile in different conditions to arrive at high yielding condition.
I hope you will be thinking of taking up all the above mentioned painful standarization step to improve your yield.
Hope this has addressed your query.
Rajkumar
Hi Giggsy,
You should play a Little bit with the conditions until you reach your best expression (with minimal amount of protein going to inclusion bodies).
I use to induce OD600 of 0.2-0.3. Then you can try IPTG from 0.1 to 1mM; 37/30/25°C; time from 3hs to 18hs, always using a non-induced culture as negative control. You can use 5-10mL cultures.
Also the culture media can be improved from LB to SOB.
Just in case, you may check if your protein is toxic for the bacteria.
Good luck
u need to optimize protein expression conditions. as suggested, try optimizing in mini preps, i mean using 5 ml cultures. following link might help :
http://www.embl.de/pepcore/pepcore_services/protein_expression/index.html
Some proteins have narrow shelf-life and auto catalysis can begin within 30-40 minutes, It is good to load them immediately and set up your reactions fast. I have the same experience while working with HBx protein of Hepatitis B virus
It may be that it expressing this protein is less than you would expect, you should try to use more micrograms of protein in your western blot or even try different concentrations, to find the optimal concentration.
Hope this helps and Good luck!
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