Fig 1: Photolysis and subsequent glutathionylation of B2M. Panel A: Representative SDS-PAGE of B2M (10 μM) incubated with or without RB (10 μM, in 10 mM phosphate buffer, pH 7.4) in the dark and then reaction with Bio-GSH. Panel B: Representative SDS-PAGE of B2M after photo-oxidation in the light and then reaction with Bio-GSH. Panel C: As panel A, but with subsequent immunoblotting and use of streptavidin-HRP antibody. Panel D: As panel B, but with subsequent immunoblotting and use of streptavidin-HRP antibody. Panel E: OD ratio (ODn min/OD0 min) of B2M monomer and dimer (0 min: white bar, 30 min: deep grey bar, 60 min: light grey bar and 90 min: black bar) from SDS-PAGE data in panel B. Panel F: OD ratio (ODn min/OD0 min) of glutathionylated B2M monomer and dimer (0 min: white bar, 30 min: deep grey bar, 60 min: light grey bar and 90 min: black bar) from immunoblotting data in panel D. Statistical differences are indicated as follows: *p < 0.05 vs. lane 4 (panels B, D; monomer); #p < 0.05 vs. lane 4 (panels B, D; dimer). Each gel represents one of three experiments carried out on independent samples. Data in panels E and F are presented as mean ± SD from three independent experiments.
Fig 2: Photo-oxidation, and also subsequent glutathionylation, of CRP and B2M decreases the affinity of these proteins towards specific antibodies as measured by ELISA. Panel A: Photo-oxidation of CRP (1.25 μM) for 5, 10 and 15 min without (white bars) or with (black bars) addition of GSH (100-fold molar excess over CRP concentration). Panel B: Photo-oxidation of B2M (10 μM) for 5, 15 and 30 min without (white bars) or with (black bars) further incubation with GSH (100-fold molar excess over B2M concentration). Statistical differences are indicated as follows: *p < 0.05 vs the t = 0 photolysis time sample without GSH; #p < 0.05 vs the t = 0 photolysis time sample with GSH. Data are presented as mean ± SD from three independent experiments.
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