Streptavidin hrp

After electrophoresis, the gels were blotted to an Immobilon-P PVDF Membrane (Merck Millipore, Burlington, MA, US) using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories) at 200 mA for 80 minutes. After blocking with 1% bovine serum albumin (BSA) (VWR, Radnor, PA, US), the membranes were probed with 1 μg/mL mAb 9G3 against the glycosylated epitope ‘KEPAPTTT’ in the lubricin mucin domain (Merck KGaA, Darmstadt, Germany¹² ) or polyclonal anti-CG antibody (Abcam, UK) 1/1000 diluted in assay buffer (1% BSA in PBS-Tween) for detection of endogenous CG in SF, followed by Horseradish Peroxidase (HRP) conjugated goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Invitrogen, USA) 1/4000 diluted in assay buffer. For the lectin staining assay, blots were first probed by 1 μg/ml biotinylated Peanut Agglutinin (PNA, Vector laboratories, CA,USA) followed by 0.2 μg/ml HRP-streptavidin (Vector laboratories). After incubations, membranes were stained by WesternBright ECL Spray (Advansta, USA) and visualized in a luminescent image analyser (LAS-4000 mini, Fujifilm, Japan). Band intensities were calculated by ImageJ 1.50i^{53 (link)}.

An in-house ELISA method was set-up and validated for measuring lubricin concentrations in SF, adapted from others^{54 (link),55 (link)}. Monoclonal antibody 9G3 (1 μg/mL in PBS) was coated on 96-well Nunc-Immuno maxisorp plates (Thermo Fisher Scientific) at 4 °C over-night. After blocking with 3% BSA in PBS + 0.05% Tween, SF samples were added as a dilution series (1/50) in assay buffer (1% BSA in PBS-Tween) and incubated for 1 hour at room temperature (RT). Bound proteins were then incubated with biotinylated PNA (Vector laboratories) (1 μg/mL, 1 hour at RT), followed by HRP-streptavidin (Vector laboratories) at 0.1 μg/mL (1 hour at RT). Between each incubation, the wells were washed three times with PBS-Tween to remove unbound reagents. Proteins were stained with 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) until blue colour was fully generated and the reaction stopped by adding 1 M H₂SO₄. Absorbances were read at 450 nm, and compared with a standard curve using recombinant lubricin (dilution series of rhPRG4 (1 mg/mL) in assay buffer). Samples were measured in duplicates and mean values are reported. The lubricin ELISA had an intra plate CV = 7.5% (n = 1 SF sample with 10 repeats) and a inter plate CV% = 7.8% (n = 1 SF sample, tested on 4 plates). Technical performance of the assay is summarized in Supplementary Table 3.