I am studying with phospho protein and ı want to detect it with flow cytometry using intracellular staining protocol. Do ı need any buffer for protect phospho proteins ?
BD are the best company in phopho-protein ICS. see attached link for further detailes
http://www.bdbiosciences.com/research/ics/techniques/index.jsp
you can try milder buffers then perm III (like perm I) especially when you proteion is in the vicinity of the cells membrane
BD are the best company in phopho-protein ICS. see attached link for further detailes
http://www.bdbiosciences.com/research/ics/techniques/index.jsp
you can try milder buffers then perm III (like perm I) especially when you proteion is in the vicinity of the cells membrane
Hi
for phospho-proteins detection in the bone marrow, I suggest you to treat the cells with FIX & PERM (Caltag). It works very well.
Good luck.
BD is a very good starting point. The buffer to use for fixation depends on your epitope. BD has some information on the Abs they use, either via the website Pinchas linked or via FACSelect (http://www.cytobank.org/facselect/). Protection of the phospho-eptiope is not required, given that you fix immediately after activation. Good luck, G.
Put your sample in ice and control if there are phosphatase and protease inhibitor inyo your buffer.
See Krutzik, P. O. & Nolan, G. P. Intracellular phospho-protein staining techniques for flow cytometry: Monitoring single cell signaling events. Cytometry 55A, 61–70 (2003).
- Use min of 106 cells
- Fix cells with 1.5% formaldehyde 10 min at RT.
- Permeabilized with ice-cold MeOH, add dropwise during low- speed vortexing.
- Incubate on ice for 10 min.
- Store at -20C or continue with the protocol
- Wash cells 3 times with PBS/1% BSA then resuspended in staining media.
- Complete according standard protocol.
Of course the success of this protocol very much depends on the choice of primary Ab.
BD is the best. I like their Lyse-Fix buffer (catalog Number
558049) in combination with ice-cold methanol. Gary Nolan's procedure works very well too (see previous comment by Vladimir).
You can of course use the perm fix kit of Becton D. The alternative which is as good and much less expansive, is to Fix the cells in 2% Paraformaldehyde, wash and add 100% cold Methanol, and store at -80°C. The cells can be stained months after.
Then wash MetOH twice at RT, and incubate in PBF with 1% FCS to rehydrate the cells, then add your Ab for one h RT, wash resuspend in PBS-FCS for FACS analysis.
I have been working with cell signaling in different cells using all phosphorylated antibodies. I would recommend cell signaling protocol for FACS, IHC and WB. Please select their (www.cellsignaling.com) antibodies, if troubles contact them.
Thanks to all for useful inputs. I have been recently using Millipore kits for detection of intracellular phosphorylated proteins (pAKT, pS6, ERK1/2) etc. They are expensive but work well.
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