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Phospho Flow Cytometry Antibody Clone Buffer Selection Guide

Each phospho specific antibody has been tested in three different flow cytometry buffer systems: IC fixation and permeabilization, Foxp3/Transcription Factor Buffer, and IC fixation Buffer/Methanol. The recommended buffer system(s) will be noted on the Technical Data Sheet for the specific antibody or in the table below. Because methanol can destroy the epitope recognized by some antibodies, ideally surface staining would be performed at the beginning of the protocol with a methanol-resistant fluorochrome (like eFluor® 450, FITC, and eFluor® 660). However, please recognize that surface staining before stimulation can have undesired effects due to activation of the cell caused by antibody binding. Therefore, we recommend staining after stimulation/treatment, fixation, and methanol treatment. Please refer to our table (Antibody Clone Performance Following Fixation / Permeabilization) that evaluates the performance of numerous clones in the methanol-based buffer system.

 

 Target  CloneCat. No. IC Fix/perm Foxp3 Buffer Se
IC Fix/Methanol
p4E-BP1 (T36/T45) V3NTY24 9107 ++ - +++
pAKT (S473) SDRNR 9715 ++ ++ +++
pBTK/ITK (Y551/Y511) M4G3LN 9015 ++
++
+++
pERK1/2 (T202/Y204) MILAN8R 9109 - - +++
pH2AX (S139) CR55T33 9865 - +++ +++
pHistone-H3 (S28) HTA28 9124 ++ + +++
pIκBα (S32/S36) RILYB3R 9035 +++ - ++
pLCK (Y505) SRRCHA 9076 ++
++
 +++
pMCL-1 (S159) RBCERNR 9038 ++ -
+++
pmTOR (S2448) MRRBY 9718 ++ ++ +++
pNFkB p65 (S529) B33B4WP 9863 +++ - +++
pp38 MAPK (T180/Y182) 4NIT4KK 9078 +++ ++ +++
pS6 Ribosomal (S235/S236) cupk43k 9007 ++ ++ +++
pSLP-76 (Y128) HNDZ55 9037 +++ - +++
pSrc (Y418) SC1T2M3 9034 ++ ++ +++
pSTAT1 (Y701) KIKSI0803 9008 -
 - +++
pSTAT3 (Y705) LUVNKLA 9033 - - +++
pSTAT4 (Y693)  4LURPIE 9044 -  -  +++
pSTAT5 (Y694)  SRBCZX 9010 -  -  +++
pSTAT6 (Y641)  CHI2S4N 9013 -  -  +++
pSYK (Y348)  moch1ct 9014  ++  ++  +++
pTyrosine  pY20 5001 ++
 ++ +++
pZAP-70/SYK Y319/Y352  n3kobu5 9006
 ++  ++  +++