Yes, cleavage of GFP can occur, either during physiological degradation/turnover of your protein or within the artificial protein-GFP linker. In some cases the GFP within a fusion protein is not accessible to the antibody, whereas the cleaved GFP is accessible, so your IP enriches for the cleaved GFP and doesn't pull down the intact fusion protein so well.

Thank you David, do you know of some ways I can get around this problem? 

Maybe try inserting a longer, flexible linker between your protein and the GFP - eg. (GGSG)x3 - a review on protein fusion linkers below:

http://www.ncbi.nlm.nih.gov/pubmed/23026637

Hi Charles，

If the band corresponding the GFP is actually the GFP band，then you can try to fuse the GFP tag to N-terminal.

However, what if the band isnot the GFP fragment? In my case, the band may be the the light chain of the antibody used for Co-IP. Even the GFP fragment is cleavaged, then the lysosome or autophagosome system will destroy it( unless the organelles' pH is enhanced by Bafilomycin or ammonia), so you cannot detected so much bright band. Perhaps you can change your GFP antibody. I used the GFP antibody(ab290,abcam) and it works well.

Best

zhiyuan

At what stage is the cleavage occurring? i.e if you take your cells and immediately boil them in SDS lysis buffer and run on a gel does the gfp remain attached or is it already cleaved off ? Likewise if you are preparing a cell extract (with protease inhibitors?) and run the sample is it cleaved? This will give you an indication as to whether your sample prepn method needs to be modified to minimise protease activity. If the cleavage occurs in the linker region then you may have to consider changing the sequence.

Hi, Charles, I am wondering whether you have figured out how to prevent the auto-cleavage/fragmentation of GFP/mCherry-fused protein. Recently I have constructed Flag-X-GFP and HA-Y-mCherry. In my western blot experiment, I can detect the full length protein and 20kDa-losing fragment both with anti-Flag and anti-HA hybridization. Indeed the linker between X and GFP is only 2 amino acids while it is 8 amino acids between Y and mCherry. I am afraid that the C-terminal fragment of GFP/mCherry is still working for their fluorescence. And it could interrupt the result of the subcellular location of X and Y. I am looking forward for your reply. Thanks.

Hi Peng Zeng Have you resolved ths issue? I am facing the very similar issue that GFP falls off from the fused protein (https://www.researchgate.net/post/A_GFP-specific_protein_truncation). I am interested in knowing if you ever came up with a solution. Thank you!

Hi, Chia-Yi Cheng ,actually I have consulted from some expertises from fluorescent field and they did not have any suggestion. And in my case the autocleavage issue is much worse with mCherry-fushed protein than GFP-fushed protein. And I just move on and ignore this issue.

Peng Zeng Thanks for sharing! I will try subcelluar fractionation and see how that goes. Thanks again.