Why the protein can not be tested? - (Apr/25/2007 )

I insert the interest protein into pEGFP-N1. Sequence result is correct, also EGFP can be watched under microscopy. However, the technian in our lab can not tested the interest protein with western blot. the reagents she used are correct. Then, what's wrong with it? Please help me. Thanks

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some possibilities; if you can watch GF of GFP-fusion protein, is there a difference in subcellular localization compared to mock GFP vector-transfected cells which means is your subcellular GF according to the awaited location of your protein of interest? (do you really express the GFP-fusion protein or only GFP w/o fusion)

or may be there are problems witth the primary Ab; is this Ab suitable for IF? so try to use it for IF... (Ab may not suitable for Wb but for IF)

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Yes, pEGFP-N1 by itself can express GFP protein. If you don't know the supposed localization of your protein and just rely on the GFP signal, you won't be able to know if your fusion protein is expressed or not under microscope (I am assuming that you did not use (or did not have) Ab for your own protein to do IF and only take a look at the transfected cells under microscope for GFP signal).

What Ab did your tech use for WB? anti-GFP or antibody against your protein? You don't see any band at all, or you only see the GFP size band? If you are using anti-GFP for WB and don't see any band, that is a little troublesome, since most of the commercial anti-GFP (that I know of) work for WB. If you used antibody against your protein and don't see any band, how about try with anti-GFP, you may have a better idea what happened

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