I have my construcsee a band at the correct molecular weight but nott in pGT7 plasmid, transformed into BL21AI when I do a test expression and check on SDS-PAGE I don't see a thick expression band. I do see a band at the correct molecular weight but I don't see the protein after purification on gel. am also getting micromolar concentrations in the end. am starting to think the problem could be in expression. my conditions are 16degrees overnight after induction with 0.04% and have also tried 0.1% and 0.01% with similar results. KIndly help.
I doubt the problem is the arabinose. You can go up to 1% but usually you get good induction even at 0.1% which you tried.
You should confirm that the band you see at the correct MW is really your protein of interest, do you only see it after induction and not in the unindexed control or in a control plasmid control?
Essentially the problem is either that you are not getting good expression, or you have a problem with your lysis and purification step.
Hi Jennifer,
I think you should do some trouble shooting.
1) Make sure that your plasmid is correct. Affinity tag in frame with your gene of interest if the tag is at N-t of the protein. Start codon in frame after cloning.
2) You mention that after purification you don't see the protein. Have you run a Western Blot on your E.coli extracts? Check if you have expression (total protein extract) and also make soluble protein fractions to make sure you will have your protein in the supernatant. If your protein is accumulated in inclusion bodies you will have to work with the insoluble fraction after lysis and centrifuging.
3) Is the thick band you see in your gel after staining with Coomassie blue stain? When inducing E.coli cells you don't usually get only one thick band in the protein profile after Coomassie, there are more proteins that are over expressed as the cell will express the machinery to import and metabolize the arabinose. If you want to know in a Coomassie if the band you see is your protein of interest, transform BL21 with an empty plasmid and use it as negative control in your expression experiments, it will allow you to know either in Coomassie stained gels or WB which bands are specific or unspecific.
4) Do you have another gene in the same plasmid that has worked previously in your lab? If so, use it as positive control in your protein expression experiments if you think the problem of is due to handling/skill issues or arabinose. If your positive control does not work for you, then you should look into the protocol/handling/reagents.
good luck .
Hello Michael J. Benedik
I only ran a denatured gel of the samples after induction, I will try include the control and see if it my protein. I was lysing at 15sec on 15sec off pulse 70% amplitude for 5 to 10 minutes. I also have tried chemical lysis Bper reagent and other sonication protocolsis . I have tried ion exchange where I still see the band but when I try IMAC I don't see it after desalting. I am suspecting the yield is too low. am getting below 0.5mg/ml when I measure the concentration after desalting.
Xi Jiang Thank you fir your suggestions, No I didn't run a western blot yet, I will try the negative control. I did sequence the plasmid and ascertained that the genes were present.
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