As a rule of thumb, Smart Tube reagents are compatible with all perm buffers that have been shown to work with formaldehyde-fixed samples, both alcohol-based and detergent based perm buffers. That said, the choice of perm buffer is typically driven by the demands of the intracellular epitopes being analyzed. Two examples:
1. Detection of phospho-activation of Stats (ie. pY701 on Stat1, pY705 on Stat3 and pY694 on Stat5) is typically vastly superior with methanol based perm buffers (including perming with simply 90% methanol + 10% PBS pre-cooled to -20C) than with detergent based perm buffers. It is thought that because the phospho-epitope being analyzed drives dimerization, the epitope is buried within the dimer and the addition of methanol (perhaps due to its mild denaturing activity) acts to expose this epitope in a way that detergent based perm buffers do not.
2. Certain detergents (saponin in particular) do not effectively permeabilize the nuclear envelope. In the case of saponin it complexes with cholesterol to form pores in the cell membrane, but because the nuclear envelope is deficient in cholesterol these saponin-based pores do not form efficiently in the nuclear envelope (mitochondrial membranes are also cholesterol-poor and present the same problem for saponin-based buffers). Many phospho-epitopes translocate to the nucleus meaning that alcohol based perm buffers would be greatly preferred over detergent based perm buffers for these analytes. Also, many of the targets of interest in cell cycle analysis translocate to the nucleus or are already in the nucleus, which is one of the reasons why for at least three decades cell cycle analysis (by flow cytometry and other methods) has overwhelmingly favored alcohol based perm buffers.
Primarily because of the points above, the majority of studies on the Publications Page of our website used alcohol based perm buffers when analyzing phospho-epitopes.
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