Coomassie plus bradford protein assay reagent

Protein extracts from PBMCs were spectrophotometrically quantified with Coomassie Plus Bradford Protein Assay Reagent (Thermo Scientific).
To evaluate the amount of HLA-C, 6 μg of protein was analyzed with MAb L31 (1 μg/ml). Protein lysates from HEK-293T and Chinese hamster ovary (CHO) cells were used as an internal standard and a negative control, respectively.
To evaluate the amount of β₂m, 4 μg of protein was immunoblotted with a rabbit anti-β₂m antibody (12 μg/ml; Abcam). HEK-293T and HEK-293T β₂m-negative (20 (link)) cell lysates were used as an internal standard and a negative control, respectively.
The α/β-tubulin antibody was used as a loading control in accordance with the manufacturer's instructions (Cell Signaling). Horseradish peroxidase-conjugated anti-mouse (Promega) or anti-rabbit (PerkinElmer) IgG was used as a secondary antibody (0.5 μg/ml). The signal was developed with the ECL Advance Western blotting detection kit (Amersham) through the AutoChemi System UVP (BioImaging System).
The amounts of HLA-C and β₂m were quantified through densitometric analysis with ImageJ software for OSX. Each sample value was normalized to its α/β-tubulin value. Differences between experiments were normalized with the same internal standard control (HEK-293T cell lysate).

Phytochelatins PC-2 (Mol. wt. = 540.6 Da) and PC-6 (Mol. wt. = 1468.6 Da) were obtained from ANASPEC (Mountain View, CA, USA). Fluorescent-labelled glutathione, PC-2 and PC-6 (conjugated with 7-methoxy coumarin) were obtained from PEPCEUTICALS Limited (Leicestershire, UK). Coomassie Plus (Bradford) Protein Assay reagent was purchased from Thermo Fisher Scientific (Rockford, USA). All other analytical grade chemicals and reagents were purchased from Sigma–Aldrich (Dorset, UK). Mice (male CD-1, 8 weeks old) were purchased from Charles River UK Ltd.