I am willing to bet that the florescence is gone. The SDS and boiling of the gfp will denature its barrel structure which gives it its fluorescent properties. Depending on your applications just use antibody against gfp. I am not sure if running a native gel preserves florescence?

Do you make a GFP fusion protein? In that case, your target protein and GFP are expressed as a new whole protein. When you do a regular SDS-PAGE, some protein might degrade and GFP might fall apart.  But the majority should still be the intact GFP fusion protein. You'll see a band with a size of 26KD + your target's size. 

If your gel is run under denaturing conditions then the signal will probably be gone. Native polyacrylamide gel electrophoresis will allow detection of GFP in the run.

Thank you all for your responses. I think we will run a western blot with a specific antibody.

Well GFP is stable in 6 M Gnd HCl and 1 % SDS page [1].

1.Mares, R. E., Meléndez-López, S. G. & Ramos, M. A. Acid-Denatured Green Fluorescent Protein (GFP) as Model Substrate to Study the Chaperone Activity of Protein Disulfide Isomerase. International Journal of Molecular Sciences 12, 4625–4636 (2011).

I prepared a lysate from K-562-GFP cell line and loaded approximately 20ug onto a SDS-PAGE gel. I loaded both boiled and unbolied samples, and was able to visualize GFP into both boiled and unboiled samples under 488 nm laser.; without any immunoassay.

Lakshmi Pillai-Kastoori In my case, I could not observe the fluorescence of GFP when I boiled the GFP-tagged proteins. The stability of the GFP tag in the boiling condition may be dependent on the characteristics of GFP-tagged proteins.

Seokho Hong for sure. I place the lysate tubes back into ice for 5 minutes before loading the gel. There may be some level of "re-naturing" going on to return the GFP into a fluorescent state. This is just a prediction.

Hi Stephane, probably you already have your anwer by now but I can tell you that with the GFP variant (the superfold) we are using in my lab the fluorescence of GFP is preserved in SDS-PAGE as long as you don't boil the samples. In this case the SDS by itself will not eliminate completely the barrell structure of GFP and will still be fluorescent. But also for this reason migration may not reflect exactly the Mr of the fusion protein. Best regards,

Hugo.

can i still see the YFP fluorescence using the following protocol

1, add 4Xsds sample buffer

2, heat at 50C for 20 min

3, run the denatured gel 5-12% for 1hr

thanks.

Hi Hai Li, that depends of how much of your YFP is still structured after 50 C 20 min. I guess you will loss some intensity but you may still see something. Be aware that folded and unfolded fluorescent protein have different migrations in SDS-PAGE.

HI, Hugo, thank you for your reply. I hope to see the YFP band in the gel but I still need to denature protein. My protein is a small membrane protein + YFP. If I do not denature protein using 50C for 20 min, then my protein would be forming as monomer, dimer, etc so it would be difficult to judge where is my protein. But I am not sure under 50C for 20 min the YFP would be still fluorescence. Thanks.