Goat anti mouse igg antibody h l hrp

Protein extracted from nematodes was electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. After the electrophoresis, the gel was transferred to a nitrocellulose membrane in a Bio-Rad (Hercules, CA) semi-dry transfer apparatus. The membrane was pre-incubated with 5% nonfat milk in TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl and 0.5% Tween 20) for 30 min at room temperature. After that, the membrane was incubated with primary antibody (Anti-phospho-p38 MAPK monoclonal antibody (1:500, Cell Signaling), or anti-Actin monoclonal antibody (1:5000, EMD Millipore)) in TBST buffer with 5% nonfat milk for 12 h at 4 °C. After washing with TBST buffer for three times (10 min each time), the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-mouse IgG antibody (H&L) [HRP] (1:10 000, GenScript)) for 1.5 h at room temperature. The membrane was developed with ECL system (Thermo Scientific, Pittsburgh, PA). Three replicates were performed.

Whole worm extracts were electrophoresed on a 10% SDS-PAGE gel and then transferred to a Nitrocellulose membrane using a Bio-Rad semi-dry transfer apparatus according to the manufacturer's protocols. After 30 min pre-incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) at room temperature, the membrane was transferred and incubated with primary antibodies in 5% nonfat milk TBST at 4°C for 12 h. Thereafter, membrane was washed three times for 10 min and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. After 1.5 hours, membrane was washed with TBST three times and developed with the ECL system (Thermo Scientific) according to the manufacturer's protocols. Anti-phospho-p38 MAPK monoclonal antibody 28B10 (1∶500) was purchased from Cell Signaling. Anti-Actin monoclonal antibody MAB1501 (1∶5,000) was purchased from EMD Millipore. The goat anti-mouse IgG antibody (H&L) [HRP] (1∶10,000) was purchased from GenScript.

The 1000,000 planarian live cells were sorted out for transfection and protein extraction. Each protein sample of cells in 96- well plates were collected in PCR tube 24 h post-transfection and homogenized in 25 μL RIPA (RIPA lysis buffer (Genstar, E122-01), 1 mM PMSF, 10 mM DTT, 1X protease inhibitor cocktail (MCE, HY-K0010)). All the protein samples were loaded for immunoblotting. The antibodies used were as follows, rabbit polyclonal RFP antibody (MBL, PM005), mouse monoclonal Flag antibody clone M2 (Sigma, F1804), NanoLuc antibody (Promega, N7000), α-tubulin antibody (GenScript, A01410), goat anti-mouse IgG antibody (H + L) HRP (GenScript, A00160), goat anti-rabbit IgG antibody (H + L) HRP (GenScript, A00098). The primary antibodies were used in 1:1000 dilution, and secondary antibodies in 1:20,000.