Horseradishperoxidase labeled goat antirabbit igg antibody

Western blots were performed to assess the amounts or integrity of human and rat CBGs
after tunicamycin treatment, deglycosylation by PNGase F or Endo H or incubations with
proteases. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Western
blots were incubated with 1:5,000 dilutions of rabbit anti-human CBG antiserum (Robinson et al. 1985 (link)) or a rabbit
anti-mouse CBG antiserum that recognizes rat CBG (Hill
et al. 2016), followed by a 1:10,000 horseradish
peroxidase-labeled goat antirabbit IgG antibody (Sigma-Aldrich). Immunoreactive CBG was
detected using ECL Prime Western Blotting Detection Reagent and an ImageQuant LAS4000 (GE
Healthcare).

To detect IgGs within raw serum, flow through, and elution fractions, SDS-Page was performed. In short, 15 µg samples were used for SDS-Page followed by subsequent Coomassie staining. Coomassie staining was performed according to manufacturer’s instructions (Thermo Fisher Scientific). Mouse IgG was loaded as positive control. For western blot analysis 10 µg samples and 100 ng agalsidase-α as positive control were blotted onto PVDF membranes. After blocking overnight in Tris buffered saline with 5% milk powder, detection was performed using an anti-AGAL antibody (ab168341, Abcam, Cambridge, UK; working concentration: 100 ng/mL) and a secondary horseradish-peroxidase-labeled goat anti-rabbit IgG antibody (12–348, Sigma-Aldrich, St. Louis, MO, USA; working concentration: 100 ng/mL).

A heat-dependent polymerization assay was used to identify RCL-cleaved CBG in plasma samples (Gardill et al. 2012 (link)). In brief, plasma was subjected to incremental increases in temperature (0.1°C/s to 70°C), prior to non-denaturing PAGE, and proteins in the electrophoresis gel were transferred to PVDF membranes using a Trans-Blot turbo transfer system (BioRad). The blots were blocked with 5% milk-PBST and incubated overnight at 4°C with a rabbit anti-human CBG antiserum (Robinson et al. 1985 (link)) diluted 1:4000 in 5% milk-PBST. Immunoreactive CBG was detected using horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:10,000; Sigma-Aldrich) for 1 h at room temperature, and ECL reagent using an ImageQuant LAS4000 (GE Healthcare). As a control in this assay, corresponding plasma samples were incubated with NE (Elastin Products Company) under conditions that specifically cleave the RCL of human CBG (Simard et al. 2014 (link)).
Denaturing SDS-PAGE (10% resolving gels) was performed on steroid-affinity gel purified CBG preparations (see above). Western blots were blocked and probed as described above. Rabbit anti-human CBG antibodies were affinity purified from antiserum (Robinson et al. 1985 (link)) using an E. coli expressed human CBG-GST fusion protein (Gardill et al. 2012 (link)) coupled to NHS-activated Sepharose (GE Healthcare).