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Imaging GFP tagged proteins, background signal due to contamination? - Imaging, background spots/speckles, contamination (Feb/15/2008 )

[attachment=4242:contam_U2OS.jpg]

Hi

I recently aquired a new osteosarcoma cell line that overexpresses a GFP tagged version of my protein of interest.

When I image them to investigate localisation of my GFP tagged protein (stably expressing...I do not transfect in this construct and no staining with primary/secondary antibodies involved) I see green dots everywhere both in cell and outside (see attached picture) which made me think the samples were contaminated (especially in those in antibiotic free media but not really in Pen Strep containing media) BUT I can't pick up anything other than the nuclei in the DAPI channel and I thought if contaminated there was a good chance of it coming up after DAPI staining. So very confused and concerned.

Any ideas?

Cheers

-Mojito-

QUOTE (Mojito @ Feb 15 2008, 04:56 PM)
[attachment=4242:contam_U2OS.jpg]

Hi

I recently aquired a new osteosarcoma cell line that overexpresses a GFP tagged version of my protein of interest.

When I image them to investigate localisation of my GFP tagged protein (stably expressing...I do not transfect in this construct and no staining with primary/secondary antibodies involved) I see green dots everywhere both in cell and outside (see attached picture) which made me think the samples were contaminated (especially in those in antibiotic free media but not really in Pen Strep containing media) BUT I can't pick up anything other than the nuclei in the DAPI channel and I thought if contaminated there was a good chance of it coming up after DAPI staining. So very confused and concerned.

Any ideas?

Cheers


sometimes, i see small, blue dots in the DAPI channel, which are most likely bacterial contamination. not quite sure about your case because your dots are green?

-qkchen-

Hi Mojito,

From what I see, it is not contamination, but your GFP seems to be concentrated in the ER of the cells. Is your protein involved in ER metabolism?

If not, it is possible that the GFP is separated from your protein of interest during post-traductional modifications. In that case, try to immunolocalize your mature protein.

Fabien

-fabien-

hi,

in my view, it should be not a mycoplasma contamination since the intracellular fluorescence is not homogeneously distributed in the cytoplasm
maybe some of the cells have burst, leaving their content on the slide...


S├ębastien

-tryptofan-

Any chance that this is a secreted protein? The spots look like vesicles and if the protein is secreted that would explain the extracellular signal.

-rkay447-

It doesn't look like contamination.

Your protein seem to be in vesicles or accumulating in small regions.

-scolix-

overexpressed protein may not distributed to its destination of function as it would harm the cells; so they precipitate or are stored in vesicles; I think that is what you are seeing; you may try a stain of vesicles (ER, Golgi, Endosomes) to show colocalization and subcellular localization

-The Bearer-