I have been working with A549 & HEK-293T cells and trying to do protein localization studies. I have conjugated my protein with His-GFP and the control is only His-GFP. I do find GFP signal in both sets. However, in my blank set (where neither Protein-HisGFP nor HisGFP is added) I find GFP signal in cytoplasm, around nucleus as I stain nucleus with DAPI.
Can anyone please suggest any info regarding auto-fluorescence in these cells or protocol for protein localization studies.
I would really appreciate your response. Thanks
Hi Apoorva - just to clarify: are you getting the "GFP" signal in the control cells stained with DAPI?
If so os it possible you are getting spectral-bleed through from the DAPI (UV/405nm) channel?
http://www.gum2012.fionastoreydesign.co.uk/Downloads/Bleed%20through%20text.pdf
In other words - do you see the "GFP" signal in unstained cells - or just in the cells stained with Dapi?
Hey Gary
Thanks for your response.
Actually I am staining all my cells with DAPI (so my control is neither protein-GFP nor GFP, just DAPI stained)
They show green fluorescence around DAPI stained nucleus. Pics attached for ur reference.
Thanks
Apoorva
Hi it does not look like bleed-through (if it was the blue and green signals would b overlapping)It is probably just auto-fluorescence (caused by pigments/proteins in the cells such as Lipofuscin).
You will have to set your laser power, gain etc. so that you no longer see this fluorescence in the green channel in your control cells (the pictures look quite "grainy" so I think you have the exposure/gain/laser quite high). Use these settings to image your GFP cells - this will give the "true" signal over this background fluorescence.
Almost all cells will "glow" in some part of the spectrum if the power/exposure is too high - this is why you need to use the negative control to set the lasers etc.
If that makes sense?
Gary
Hey Gary
Yea i understand ur point. I will to reduce the exposure settings and check for true gfp and negative control cells.
Thanks a lot for ur suggesstion.
Apoorva
DAPI has a very broad emmission spectra and so you will see DAPI signal in the GFP channel. It's a terrible dye to use if you have other fluorophores in there. Try DRAQ5, or the whole POPO, BOBO, YOYO, TOTO, JOJO, TO-PRO range of dyes which may be better alternatives to stain DNA for this application.
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