Checking For Protein Expression - (Aug/17/2009 )

Hi All,
I tried expressing a protein on pGEX vector with an His-Tag and found no bands on my western blot.
My supervisor told me to check for protein expression first to see if the protein is actually induced or not.
I used IPTG for expression so I'll will have one with IPTG induced and other without any IPTG. I will pellet down the cells and add SDS-PAGE loading buffer and boil to lyse the cells and run it.
The other suggest was to compare just the bacteria (BL12(DE3) lysogen) without the plasmid grown in LB plate without any resistance and the one with the plamisd grown in LB plates with AMP. I don't really get what the purpose of this is and how to carry it out....
Can somebody please help me.

Thanks.

bkool4ya on Aug 17 2009, 09:38 AM said:

To test for protein expression, you simply do the following, take 40 ul of uninduced bacteria and add 40 ul of 2x SDS sample buffer, boil at 95 C heat block for 5 minutes, load on to SDS gel. Do the same for induced bacteria and run them side by side on SDS gel.

Hmm. Were you sure that it wasn't 'no plasmid; grow in LB with/without AMP'?

Sometimes transformation does not work properly. When it doesn't, the cells should not grow on agar plates that have antibiotic on them (as the plasmid carries specific enzymes which breakdown the antibiotic and allow the cells to grow).

Possibilities spring to mind:
1. Antibiotic isn't present
2. The transformation didn't work
3. The inducer is at the wrong concentration / has been stored incorrectly and degraded

However, many antibiotics also degrade quickly. For example, tetracycline is light sensitive. Ampicillin and Kanamycin breaks down after a while when in solution or gel (this is why they usually come in powder form). Adding antibiotic while the agar is still too hot can also cause them to break down.

If the cells with no plasmid grow on both (agar + AMP) and (agar + no antibiotic), then you know that the antibiotic has broken down, isn't present or isn't at the right concentration to kill off the cells which lack the plasmid.

If the antibiotics are fine, then the problem could be that the transformation wasn't ok. Check that the cells are competent (treated so that they take up DNA easily). Heat-shock works, but I've heard that electroporation is more efficient at transformation...

Have you determined yet whether the protein is expressed as suggested (i.e., by comparing the total protein profile of the isogenic parent and the target producing strain on SDS-PAGE or immunoblot )? If the protein is not accumulating, I suggest using an in vitro transcription-translation kit to diagnose if the problem lies with the expression vector construct or the cell's inability to accumulate the target protein. Should you not recover any product in vitro, then consider a different vector design.