Trypsin lysc protease mixture

The N terminal Flag tagged PGRMC1 plasmid was transfected into HEK293 and HepG2 cells and the cells were serum starved for 24 hours. The cells were lysed using 1× phosphate buffered saline (PBS) supplemented with 1% IGEPAL-CA630 (Sigma, Cat. No: I8896) and 1.5% n-Dodecyl β-D-maltoside (Sigma, Cat. No: D4641). The Flag-PGRMC1 was then pulled from total cellular lysates using Bs3-conjugated (bissulfosuccinimidyl-suberate, ThermoFisher, Cat. No-A39266) as well as unconjugated Dynabeads-protein G-anti-Flag antibody. Dynabeads-protein G (ThermoFisher, Cat. No: 10004D) was used as a negative control in the immunoprecipitation. The immunoprecipitated Flag-PGRMC1 complex was denatured, reduced, alkylated and precipitated. The precipitated proteins were dissolved in 6 M urea, with volume adjusted using 20 mM Tris buffer (pH 8.0), and digested overnight at 37°C on a shaking platform using a Trypsin/Lys-C protease mixture (Promega, Cat. No: V5071). The digested peptides were desalted using Pierce C-18 100 µl tips (ThermoFisher Scienti c, Cat. No: 87784) and analyzed by tandem mass spectrometry (MS) analysis using the AB SCIEX TripleTOF™ 5600 System (Applied Biosystems/MDS Sciex, Foster City, CA) at the Manitoba Centre for Proteomics and Systems Biology, University of Manitoba.