Strange phenomenon with western blot - Double bands (Jan/04/2007 )

Hi everyone,

A few weeks ago, i created a GFP fusion protein.
To test on western blot if the fusion protein was succesfull, I transfected 4 different types of mine GFP-tagged protein (130 KDa) a free GFP control and a non transfected control.
After blotting with anti-GFP, I saw a band at ~160 KDa by the fusion proteins and no free GFP, By the GFP control arised a band from ~30 KDa (Free GFP). The non-transfected control was, as expected empty.

After sripping my blot, I incubated the blot with anti-actin (for loading control) and the upper part of the blot with a anti-body against my protein.
The loading control was OK, every lane had the same amount of protein.
But, I saw a strange phenomenon at the blot of mine protein of interest; I saw that the band of mine fusion protein of interest was higher then the control (~160 kDa, difference of ~30 KDa with the control), but the endogenous levels (~130 KDa) of mine protein of interest were also much higher then the control.

Is it possible that when I transfect my protein-fusion protein into cells (HEK), there arises as artifact an upregulation of my protein of interest? Normally, it should not be happening that GFP is disconnected from my protein of interest, and I also did not see any free GFP...
Because my protein of interest is a peptidase, could it be that my protein degradates GFP?

I hope you understand my question, it is a little bit hard to explain...

Are you using the same concentration of your antibody to your protein of interest and your antibody to GFP? They might just need to be optimized. Maybe your protein of interest antibody is "better", meaning that you need less and in your experiment you used too much. That would cause it to bind to everything in your prep, including any free GFP. Maybe your anti-GFP antibody was used in too low a concentration so that you didn't see all of the GFP present, free and bound to your protein.

Or maybe during your stripping process, you broke bonds between your anti-GFP and the GFP-protein of interest that caused the GFP itself to break loose and then bind back to the membrane by itself? Which shouldn't happen because you block, but I am just throwing it out there for fun.

I use two controls to check en endogenous levels pf my peptidase, these levels are the same as the GFP control, while the endogenous levels of my fusion-proteins are much higher, I think that er there are two possibilities; 1. The peptidase cuts its own GFP tail off and 2. Because of over-expression of the peptidase, the endogenous levels are also upregulated.