western blotting, (GFP is there - But where's the protein I want?) (Jul/29/2004 )

Hi:

I have inserted my DNA fragments (500bp and another is 1500bp) into an AAV-IRES-hrGFP vector. This vector contains DNA sequences encoding for green fluorescent protein which will be used as an indicator of expression of my inserted fragment. After transfection with these two kinds of plasmid, I want to find out if these plasmids can be expressed in cells (293T).

Green fluorescent can be seen easily in the cultured cells. This usually means that my fragment is expressed together with GFP separately. (In the same mRNA but the proteins are separated with each other)

Then I test the cell lysate by western blotting. One is ok, but I canot find the other protein by western blotting.

I don¡¦t know the reason.

The structure of the plasmids is correct. The rate of the cultured cells which express GFP is nearly 80%. And I get good results in positive and negative control in western blotting.

Could it be out of frame?
Do you have a positive control for the antibody?

Have you eliminated/deleted/mutated your stopcodon in the fragment you are inserting?
Can you detect ANY GFP in your western (i.e. degraded or pure GFP; I guess you should, if you can see the GFP in the microscope..) or is there nothing in the lane??

Iam having the same problem with IRES vectors. I have a vector with the same set up as your GFP vector (promoter-GOI-IRES-GFP) as well as another vector with the NEO resistance gene in place of the GFP (promoter-GOI-IRES-NEO). In both cases I have found high percentages of glowing green cells not producing my protein of interest, or cell lines derived form the NEO vector living fine in high doses of G418 which also do not express my POI.

Technically this is improbable since they are driven off the same promoter and are on the same mRNA, but even the litereature does not guantee 100% efficiency. The cells are able to preferancialymake the second protein without making the first.

One thing you might check is if you havea good Kozac sequence before your start ATG to help initialize traslation.

In side by side experiments (multiple) I have found pCDNA3.1 to be far superior to pCI-NEO, pIRES-NEO and pIRES-EGFP vectors for mammalian expression.

(this is certainly NOT an advertisement, just my experience