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™ 450FITC, 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/permFoxp3 buffer set
IC fix/methanol
p4E-BP1 (T36/T45)V3NTY249107++-+++
pAKT (S473)SDRNR9715+++++++
pBTK/ITK (Y551/Y511)M4G3LN9015++
++
+++
pERK1/2 (T202/Y204)MILAN8R9109--+++
pH2AX (S139)CR55T339865-++++++
pHistone-H3 (S28)HTA289124++++++
pIκBα (S32/S36)RILYB3R9035+++-++
pLCK (Y505)SRRCHA9076++
++
 +++
pMCL-1 (S159)RBCERNR9038++-
+++
pmTOR (S2448)MRRBY9718+++++++
pNFkB p65 (S529)B33B4WP9863+++-+++
pp38 MAPK (T180/Y182)4NIT4KK9078++++++++
pS6 Ribosomal (S235/S236)cupk43k9007+++++++
pSLP-76 (Y128)HNDZ559037+++-+++
pSrc (Y418)SC1T2M39034+++++++
pSTAT1 (Y701)KIKSI08039008-
 -+++
pSTAT3 (Y705)LUVNKLA9033--+++
pSTAT4 (Y693) 4LURPIE9044- - +++
pSTAT5 (Y694) SRBCZX9010- - +++
pSTAT6 (Y641) CHI2S4N9013- - +++
pSYK (Y348) moch1ct9014 ++ ++ +++
pTyrosine pY205001++
 +++++
pZAP-70/SYK Y319/Y352 n3kobu59006
 ++ ++ +++