Tgx criterion gradient sds polyacrylamide gels

Muscle lysate protein (80 µg total protein) was separated on 4–20% TGX Criterion gradient SDS polyacrylamide gels (Bio-Rad, Berkeley, CA) and processed for mass spectrometry, as previously described (12 (link)). Mass spectrometry (liquid chromatography–electrospray ionization–tandem mass spectrometry [LC-ESI-MS/MS]) was performed on a Thermo Electron (San Jose, CA) Orbitrap Elite Velos Pro fitted with an EASY source (Thermo Electron, San Jose, CA). NanoLC was performed using a DIONEX/Thermo NCS-3500RS UltiMate 3000 with an EASY Spray column (Thermo Electron, San Jose, CA; 50 cm × 75 μm inner diameter, packed with PepMap RSLC C18 material, 2 μm). A “top 15” data-dependent MS/MS analysis was performed (acquisition of a full-scan spectrum followed by collision-induced dissociation mass spectra of the 15 most abundant ions in the survey scan).

Muscle protein lysates (20–40 µg total protein) were resolved by TGX Criterion gradient SDS polyacrylamide gels (Bio-Rad, Berkeley, CA), transferred to polyvinylidene fluoride membrane, and probed using various antibodies. ImageJ was used for quantification of the blot (http://imagej.nih.gov/ij/index.html).