Phospho tyrosine mouse mab p tyr 100

Naïve and vemurafenib resistant 1205Lu cell lines were each grown in ten 15 cm tissue culture dishes. Cells were grown to ~70% confluency prior to washing each dish with 10ml of ice cold PBS + 1mM orthovanadate (Sigma Aldrich, St. Louis, MO). Cells were then lysed according to the manufacturer’s instructions for the Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Cell Signaling, Beverley, MA). Lysed proteins were reduced and alkylated prior to proteolytic digestion and phosphorylated tyrosine containing peptides generated from tryptic digestion were enriched with antibody-based (P-Tyr-100) immunoprecipitation. Flow through from the immunoprecipitation was then further enriched for phosphorylated serines and threonines by strong cation exchange peptide fractionation (SCX) and immobilized metal affinity chromatography (IMAC). The enriched phospho tyrosine, serine and threonine fractions were then subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and the resultant tandem mass spectra was searched against SEQUEST and MASCOT databases to identify phosphoproteins. To calculate relative phospho-signal intensities, label-free protein quantification of the mass spectrometry data was analyzed using MaxQuant version 1.2.2.5 (18 (link)).