Horseradish peroxidase conjugated rabbit anti human igg

Purified GlpQ (500 ng) was electrophoresed on each replicate lane of a precast mini 4%�?"20% sodium dodecyl sulfate�?"polyacrylamide gel electrophoresis gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to a nitrocellulose membrane using the Bio-Rad MiniTrans Blot Cell (Bio-Rad Laboratories). Replicate strips containing rGlpQ were blocked overnight at 4A�C in PBS (pH 7.2)/5% dried milk/0.05% Tween 20. The blocked strips were then individually incubated with human serum at a 1:250 dilution at room temperature in PBS (pH 7.2)/2.5% dried milk/0.05% Tween 20 for 1 h. The strips were then washed 3 times and incubated for 1 h with horseradish peroxidase�?"conjugated rabbit anti-human IgG (Sigma-Aldrich, St. Louis, MO, USA) or with horseradish peroxidase�?"conjugated goat anti-human IgM (Invitrogen) at a 1:5,000 dilution in PBS (pH 7.2)/2.5% dried milk/0.05% Tween 20. Bound antibodies were detected by using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Serum from �%^10% of the study participants reacted to a �%^55-kDa band, presumably a trace contaminant copurified with the rGlpQ generated in a bacterial expression system. Samples with a 39-kDa band corresponding to GlpQ on positive control mouse serum samples were considered GlpQ antibody�?"positive (Figure 1).