TRRAP-interacting proteins during heat shock

Project description: HeLa cells and cells stably expressing hTRRAP-3xFlAG were subjected to mass spectrometry (MS) analysis. Briefly, Cells were treated or untreated with heat shock at 42°C for 30 min, and TRRAP was immunoprecipitated from these cell extracts with 60 ul anti-FLAG M2 affinity gels by rotating at 4°C for 3 h. Immunoprecipitated sample was eluted by 40 ul FLAG peptide at 4°C for 30 min. Protein samples were loaded on 12% SDS-PAGE for preparation of gel slices. Gel bands were cut-out and subjected to in-gel digestion with trypsin. Resulting peptides were dissolved in a solution containing 0.1% trifluoroacetic acid and 2% acetonitrile and analyzed by an LTQ Orbitrap Velos Pro mass spectrometer coupled with a nanoLC instrument and HTC-PAL autosampler.