Anti mouse igg whole molecule alkaline phosphatase antibody

Ten ng of recombinant H4 HA and H11 HA (negative control) prepared in PBS were mixed with equal volume of 2x Laemmli loading buffer (Bio-Rad) supplemented with 2% beta-mercaptoethanol (BME) and heated at 100°C for 15–20 min. Samples were then run on sodium dodecyl sulphate (SDS) polyacrylamide gels (5–20% gradient; Bio-Rad) and later transferred onto nitrocellulose membranes. The remaining procedure was directly adapted from a published protocol [55 (link)]. Membranes were blocked with 3% non-fat milk in TPBS and then stained with 30 μg/mL of each antibody prepared in 1% non-fat milk in TPBS. Membranes were then washed three times with TPBS and stained with anti-mouse IgG (whole molecule)–alkaline phosphatase (AP) antibody produced in goat (Sigma-Aldrich). Reactivity was visualized by treatment of membranes with development solution prepared from AP conjugate substrate kit (Bio-Rad).

Ten ng of recombinant H4 HA and H11 HA (negative control) prepared in PBS were mixed with equal volume of 2x Laemmli loading buffer (Bio-Rad) supplemented with 2% beta-mercaptoethanol (BME) and heated at 100°C for 15–20 min. Samples were then run on sodium dodecyl sulphate (SDS) polyacrylamide gels (5–20% gradient; Bio-Rad) and later transferred onto nitrocellulose membranes. The remaining procedure was directly adapted from a published protocol [55 (link)]. Membranes were blocked with 3% non-fat milk in TPBS and then stained with 30 μg/mL of each antibody prepared in 1% non-fat milk in TPBS. Membranes were then washed three times with TPBS and stained with anti-mouse IgG (whole molecule)–alkaline phosphatase (AP) antibody produced in goat (Sigma-Aldrich). Reactivity was visualized by treatment of membranes with development solution prepared from AP conjugate substrate kit (Bio-Rad).

The samples were heated in 2× Laemmli buffer with 2% beta-mercaptoethanol (BME) at 100°C for 20 min and run on polyacrylamide gels (5% to 20% gradient; Bio-Rad). Gels were stained with SimplyBlue SafeStain (Invitrogen) for an hour and then destained in water for another hour. For Western blot assays, SDS-PAGE was performed with the sGPCe side by side with an irrelevant control protein (recombinant influenza A virus H7 protein). The gels were then transferred onto nitrocellulose membranes according to a previously described protocol (39 (link)). Each monoclonal antibody was used for primary staining at 30 µg/ml, and the secondary staining was performed using anti-mouse IgG (whole molecule)–alkaline phosphatase (AP) antibody produced in goat (Sigma-Aldrich) at a dilution of 1:1,000. The membrane was developed using an AP conjugate substrate kit, catalog no. 1706432 (Bio-Rad). To ensure that the proteins were transferred onto the nitrocellulose membrane, an anti-His monoclonal antibody (TaKaRa Bio Company) was used as a positive control, detecting a hexahistidine tag on the GPCs and the control protein.