I reckon you refer to SDS-Page, anyway not all protein express well or express at all in ecoli. Sometime the expressed protein is toxic to cells and you have only minimal expression that you won't see on an SDS-Page gel but u can run a western blot and bind a specific antibody, maybe if your protein has an His-tag you can detect that. Another thing to check is if the protein is insoluble and it goes in the inclusion bodies, in that case you need to solubilise the pellet. Good Luck.

Did you sequence your DNA-vector before transformation? I also had a positive colony-PCR one time but after sequencing the vector did not contain my gene of interest.

Marcus..i did colony pcr and restriction digestion to confirm the presence of my gene of interest.

Dear Abhilek, If you are confirming your gene of interest and you are getting your results as per expectation, then you should check your protein of interest which you are trying to express is with suitable host , If it is reported anywhere , then check whether your protein of interest is within inclusion bodies or not, if yes then load on to the SDS-PAGE and analyse the pellet fraction by solubilizing in high denaturing condition

I agree with Domenico. Collect cells before and after induction, and after lysis, load (separately) the supernantants and the pellets of both the induced and non induced E. coli. There is a good chance that your protein is in the insoluble fraction.

good luck,

Patrick

I agree with Patrick, that will clear all the doubts. But, if your protein is eukaryotic and having glycosylation site, the chances of getting your expressed protein of interest is in inclusion bodies.

Patrick's suggestion is very sensible, and Mukesh rightly points out that eukaryotic proteins often fail to express solubly in E. coli. Another suggestion would be to run a western blot and use anti-His tag antibodies to specifically identify your protein of interest. This helps if your expression is low, or if the protein is of a similar molecular weight to an abundant E. coli protein.

Obviously this will only work if your protein is His tagged, but I guess that is likely if you are using pET28. Many modern commercial SDS-PAGE ladders include His tagged proteins (e.g. my favourite, https://www.thermofisher.com/order/catalog/product/26634, has several that respond well with a His-tag), so you might not need a separate ladder - ask the manufacturers of your standard ladder! There is a good chance that someone else in your department will have all the equipment/reagents needed.

In addition to previous comments, some proteins are toxic to the bacterial cell. In that case, you might not see any prominent band for your protein in the SDS gel. If this is the case, try active site mutant of this protein.