Rabbit hrp conjugated secondary antibodies

Cell lysate was extracted from FFPE tissue samples using a Qproteome FFPE Tissue Kit (Qiagen). 15 μg of total protein from each sample was run on 4–12% bis-tris precast polyacrylamide gel (Thermo Fisher Scientific), transferred to PVDF membrane using a wet system and probed overnight with primary antibody. The following primary antibodies were used: rabbit polyclonal anti-Histone H3 antibody (Abcam, Cambridge, MA), rabbit monoclonal anti-histone H3 K27M (RevMAb Biosciences) and rabbit monoclonal anti-trimethyl-histone H3 (Lys27) antibody (Merck Millipore). The blot was then probed with the rabbit HRP-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK). Membranes were revealed using chemiluminescent detection reagent (GE Healthcare) and enhanced signal was detected in CCD imaging system. Protein extracted from the FFPE tumor sample of H3F3A wild-type glioma (IDH1 mutated) case was used as a negative control for H3 K27M and as a positive control for H3K27me3 [19 (link)].

FN, EMILIN1 or gC1q and its mutated variants were incubated with the recombinant MMPs or NE at different molar enzyme/substrate ratios for different times as indicated at 37 °C. The digestion with MMP-3, MMP-9, MT1-MMP and collagenase was carried out in a buffer containing CaCl₂ (10 mM for MMP-9 and MT1-MMP; 5 mM for MMP-3), 150 mM NaCl, 50 mM Tris, pH 7.5. The solution for NE digestion contained 10 mM CaCl₂, whereas for PR3 a 150 mM NaCl, 50 mM Tris, pH 7.5 buffer was used. Controls were incubated in the appropriate buffer without enzymes. After the indicated times, reactions were stopped by adding 5 × Laemmli buffer, loaded onto 4–20% polyacrylamide gel, and stained in a 0.05% wt/vol Coomassie G-250, 5% v/v glacial acetic acid solution to monitor substrate degradation. In some experiments we analyzed fragmentation pattern by western blotting technique. The nitrocellulose membranes were saturated with TBS buffer (20 mM Tris and 0.15 M NaCl) containing 0.1% Tween-20 (TBST) and 5% non-fat dry milk for 1 h at room temperature and then incubated at 4 °C overnight with primary antibodies against EMILIN1 or gC1q (As556 and AP As556, respectively). After extensive washing in TBST, the membranes were incubated with rabbit HRP-conjugated secondary antibodies (Amersham, GE Healthcare) and then revealed with the ECL Plus chemiluminescence kit (Amersham, GE Healthcare).