In one of my experiments, I used EGFP as control and I see some localization of EGFP on the actin cortex/stress fibres. Is this some non-specific binding of GFP antibody or some population of EGFP actually localizes with actin?
I did IFA on the non-transfected cells with the same antibody and do not see such binding. However, I do see some non-specific signals from the nucleus. I wanted to confirm if it is natural to see such localization of EGFP on actin, and binding of GFP antibodies in the nuclear region. Can anyone suggest a good antibody against GFP?
Images for both are attached.
Hi Harsh,
check this paper:
Article Nuclear localization of enhanced green fluorescent protein h...
Basically authors state that eGFP can be translocated to nucleus when expressed in mammalian cells.
Microscopy settings (laser parameters) are very important to determine if the signals you are looking at are real or artifacts due to parameters. In the case of the non transfected cells, the importance of having that control is to set the parameters in order to detect signal above that background signal. If you force the parameters to see some GFP emission, of course you would see some signal coming from it. Once you establish the limits based on the non-transfected cells, I think the pictures you took with the transfected cells will look better.
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