I created a GFP-fused mutant of my favorite transcription factor and expressed it in 293T cells. The mutation is in the putative DNA binding domain. It is not co-localizing with regions of dense chromatin. The wild type protein actually appears to be excluded from these structures. Any insight would be great!
Nuclear bodies or nucleoli? It looks more like nucleoli according to google image search.
Hi Laura,
since the nucleoli don't seem to give GFP fluorescence I would guess that your GFP-fusion protein localizes to cajal bodies.
Co-staining with fibrillarin and/or p80/coilin might give a definitve answer.
Best
Kai
First of all, if you have a strong promoter GFP can cause aggregation as an artifact. Is there an antibody available for your favorite transcription factor? If so, staining endogenous protein with it would be a nice complement to this data to see where it goes under normal circumstances. Otherwise, it could be any number of nuclear bodies, hard to say given the image. Like Kai suggested above, co-staining with markers of other nuclear bodies (ex. Cajal, PML bodies, speckles, paraspeckles, Gems, etc...) might give you a better idea. I agree that it does not look like it is in the nucleoli, as they are clearly visible in the image and do not contain the GFP. Also, there are many more foci than the typical number of nucleoli in 293s. As an alternative approach, if you've got the resources, immunoprecipitation (anti-GFP) followed by mass spec (typical proteomics pulldown) might provide some insight into the proteins it associates with and those that it is associating with in the pictured bodies.
I agree Dr. Simko. Strong expression of gfp-fusion protein occasionally results in non-specific aggregates. In addition, location of gfp (N- or C-terminus) sometimes misleads the localization of your target protein. Using protein-specific antibody or alternative tag (such as flag) will provides supporting data.
Thank you for your responses. The GFP is under the CMV promoter so it is strong, however, the wild type version of this protein (which is two amino acids different than the one imaged above) does not aggregate. It is exclusively nuclear and, in fact, it is actually excluded from these foci. I'm not sure it is aggregation but it is certainly possible. As for staining for the endogenous protein, it isn't expressed in 293T cells. I think I will try co-staining for other markers as suggested. The IP is a great suggestion. And so is the alternative tag. I will look in to doing both. I am just baffled by why a DNA-binding mutant that otherwise associates with active chromatin would be relocalized or sequestered into these foci. Thank you again for your suggestions!
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology