Does anybody have any experience with quantifying nuclear EGFR (or any other nuclear protein) using FACS analysis? The EGR is normally, and far more abundantly located on the plasma membrane. I am interested in quantifying the smaller proportion of EGFR that localizes to (within) the nucleus. Any help is welcome!
Tricky. My guess is that you would have to have an unlabelled antibody to block the cell surface expressed then permeabilise the cell and then detect. I imagine this would still be tricky to get working accurately and quantitatively as your surface blocking would need to be perfect. you might be better having a fixed number of cells and run the whole lysate on a gel next to a nuclear extract from the same number and do a western- even that isn't perfect as you would lose some protein through the extraction step but might work reasonably well. Hope these give food for thought.
or could I sort nuclei using an EGFR antibody? Can nuclei be permeabilised?
Protocols exist to isolate nuclei - some people at Centenary did this years ago for division tracking using CFSE. Not sure if eGFP would survive that same protocol but would be worth a shot.
http://onlinelibrary.wiley.com/doi/10.1002/1097-0320(20000701)40:3%3C230::AID-CYTO8%3E3.0.CO;2-E/full
This paper shows internal detection of FOXP3 quite nicely and we have done similar in our lab- it will pick up cytoplasmic of course but in your case this is probably no bad thing as the EGFR is heading to the nucleus anyway:
http://jid.oxfordjournals.org/content/193/11/1557.full
I think it would be too cumbersome to isolate nuclei and look at EGF expression. The simplest way is to do immunofluorescent microscopy. Try and read upon literature on compounds or mutants that trap EGF in the nucleus. There is a lot out there on this. Then you can just grow your cells on a slide, fix, permeabilise, stain and look at nuclear expression. For quantification just use ImageJ.
Hi i agree concerning the suggestion to use microscopy as alternative to facs but you dont have to do the manualy. Maybe you can find someone who runs a high content screening microscope. The idea behind this techique is to combine the advantages of facs(High throughput, gating...) with these of microscopy(morphological information). In general, many pictures are taken by an fully automated microscope and then processed by a software which can learn to identify cells and measure properties like fluorescense intensity, morphology and much more. The advantage is that the sorfware can easily identify nuclei and you can restrict the measurement only to this region of interest. After you have defined the rules, the software can identify and measure 1000s of cells in allmost no time automaticalyIn the end you have Data on many cells which you can gate like you would do usung a facs.
You can also use Image Stream (if you know someone which has one) which is combining FACS and fluorescent microscopy. There you can acquire 50000 events or even more.
Hi, we are currently developing a technology that might help you with this problem (www.chipcytometry.com) Can you tell me the mAb you are using and the celltype you would like to look at? Then we could test how quantitative results would look like using our cytometer. I think we would use DAPI or Hoechst to identify the nuclei and calculate a mean nuclear fluorescence for EGFR...
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