I have seen a few reference for bacterially isolated gfp protein on native PAGE however I work with mammalian cells. If somebody is doing the similar kind of work, can they share the protocol?
Meenu, you will need to check the emission wavelength of your UV transilluminator. GFP has an excitation peak at 395 nm so a short-wave UV transilluminator (usu 254 nm) probably won't work for GFP. However a long-wave UV lamp at 365 nm might do the trick. I have not tried to visualize GFP on an SDS-polyacrylamide gel though.
Interesting question, I have worked with GFP fusion proteins during my thesis yet never tried it. I think several parameters should be checked first:
- is it GFP or eGFP?
- Check excitation/emission spectra
- Make sure protein extraction method does not abolish possibly important secondary structures needed for GFP to fluoresce.
Good luck
Inspired by this post:
https://www.researchgate.net/post/Membrane_Protein_Western_Blot
I tried the same thing with proteins overexpressed in S2 and HEK293 cells. Just lyse your cells as usual (I use NaCl/Tris w/ TritonX-100 and EDTA), get rid of insoluble material (12.000g/20 min/4 deg Celcius). At this stage you can check if the lysis worked and if you have enough fluorescence with a fluorescent binocular microscope, just for your sample next to a tube with lysis buffer only and you should see fluorescence well above background fluorescence. Then denature in sample buffer (the GFP really doesn't care) and run on a regular polyacrylamide gel.
For the imaging I use a LAS3000 image reader with either the GFP and SyBr-Green filter set. Bands are clearly visible but the PA gels give quite some background. The only difference to a regular WB is to load more sample, depending on your expression level. I'd try to lyse the cells in less lysis buffer and run different amounts.
Hey Peters! Thanx for your reply..Have you tried to visualize the PA gel on regular UV transilluminator? Since we don,t have Laser reader in our lab..
@Meenu: Yes, I tried to visualize it with a regular UV table which we use for visualizing ethidium bromide stained gels, I couldn't see anything. Perhaps with an appropriate filter set it should be possible but I didn't try. I also tried to see it with the fluorescent binocular microscope which didn't really work for that purpose because it is impossible to see the whole gel at once. Btw, the LAS3000 imager is LED based, much cheaper than a laser.
@Elia: The beauty of GFP (one of its hundred beauties) is its stability, I denatured my samples in SDS/DTT at 60 degree C, ran them on a gel and the fluorescence wasn't abolished. The structure of GFP is extremely stable.
Meenu, you will need to check the emission wavelength of your UV transilluminator. GFP has an excitation peak at 395 nm so a short-wave UV transilluminator (usu 254 nm) probably won't work for GFP. However a long-wave UV lamp at 365 nm might do the trick. I have not tried to visualize GFP on an SDS-polyacrylamide gel though.
Thanks for your answers. I'll try with our 302 nm UV transilluminator, then see what happens...
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