I have tried to detect phospho-MLKL in a Western Blot using the positive control cells L929 treated with 100 ng/ml TNF alpha, 100 nM Smac mimetic and 30 uM Z-VAD for 6h and then harvested.
However I have not been able to detect the protein using the commercial antibody Anti-MLKL (phospho S345) (ab196436) from Abcam.
Does anybody know the protocol conditions for the sample preparation and protein analysis needed to be able to detect this phosphorylated protein?
Thank you in advance.
See this paper for details on Ab: http://advances.sciencemag.org/content/2/7/e1600224.full
I have copy-pasted the protocol. I remember that we always had to use M2 buffer for detection of MLKL.
For analysis of MLKL protein, cells were lysed in ice-cold M2 lysis buffer [50 mM NaF, 20 mM tris (pH 7.0), 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, and 3 mM EGTA, supplemented with Roche protease and phosphatase inhibitor cocktails] for 30 min before centrifugation to pellet insoluble component as described (47).
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