Pierce bca assay

CME was prepared from adult rat brains as described by us previously [21 (link)]. Briefly, brains were homogenised in sucrose/EDTA (Sigma, Poole, UK) and debris was clarified by centrifugation. The supernatant was layered on 0.9 M sucrose and centrifuged at 20,000 g for 60 min. The white material at the interface was collected and purified in 0.32 M sucrose solution and the white pellet was collected and freeze-dried overnight. The protein content of the CME was determined using the Pierce BCA assay (#5000001; BioRad, Watford, UK) and the content of myelin-derived axon growth inhibitory molecules such as myelin basic protein (#MCA409S; MBP; Serotec, Oxford, UK; 1:500 dilution), Nogo-A (#AB588; Sigma; 1:500 dilution), myelin-associated glycoprotein (#MAB1567; MAG; Sigma; 1:500) and the CSPG brevican (#SC-135849; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:500 dilution) was determined by Western blot, as described below.

Samples were prepared by homogenizing ~ 50 mg of tissue in ~ 550ul (11ul:1 mg) RIPA for skeletal muscle and ~ 500ul (10ug:1 mg) RIPA for liver. RIPA contained protease and phosphatase inhibitor cocktail (Pierce). Tissue samples homogenized using a TissueLyserII (Qiagen) for 2 min at 30 Hz for skeletal muscle and 1 min at 30 Hz for liver. Supernatant after centrifugation was collected and protein measured using Pierce BCA assay (Bio-Rad).
Blotting was performed with Criterion TGX gels (Bio-Rad). Gels were transferred to PVDF membranes (Bio-Rad) and total protein measured with Ponceau S (Sigma) staining imaged with a Perfection V39 flatbed scanner (Epson). Membranes were blocked with 10% non-fat dry milk or 2% BSA in TBST. Membranes were incubated with primary antibody overnight at 4 °C with agitation: Akt (Cell Signaling, 9272), SOD2 (AbCam, ab13533), GPX4 (Santa Cruz Biotechnology, sc-27529), pAkt(S473) (Cell Signaing, 9271), GPX1 (AbCam, ab22604), HNE (Alpha Diagnostic, HNE11-S). Membranes were washed with TBST and incubated with HRP secondary (Santa Cruz Biotechnology) for 1 h at room temperature. Membranes were then washed and developed with Pierce ECL Plus Western Blotting Substrate (ThermoFisher). Membranes were imaged on a Typhoon FLA 7000 (Amersham). All quantifications were performed in ImageStudioLite (LI-Cor).