Horseradish peroxidase hrp linked anti mouse igg

Total protein (20 µg) extracted from leaf tissue (1 g) was separated by 12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on to a nitrocellulose membrane. The membrane was blocked overnight in 1X TBST buffer (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) with fat free milk (5%) at 4 °C. Blots were incubated with anti-GFP mouse IgG1κ antibody (Roche, Switzerland), and then with horseradish peroxidase (HRP)-linked anti-mouse IgG (GE Healthcare). Cross-reacting bands were detected using enhanced chemiluminescence immunoblotting detection reagents (GE Healthcare) and a chemiluminescent analyser, LAS3000 (GE Healthcare).

Ten micrograms of total cellular protein was separated by 10% SDS-PAGE and transferred to a Hybond P nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% skim milk in TBS, incubated with the M2 anti-FLAG mouse monoclonal antibody in TBS, washed with TBS, and incubated with horseradish peroxidase (HRP)-linked anti-mouse IgG (GE Healthcare) in TBS. After being washed, the membranes were immersed in the peroxidase stain kit solution (Nacalai Tesque Inc.).

For analysis of PLCγ1 phosphorylation, C2C12 cells or MC3T3-E1 cells were stimulated with LAMZ (10 μmol·L⁻¹) after serum starvation (6 hours). The total proteins of these cells were extracted with lysis buffer containing Triton X–100 (1%), NaCl (150 mmol·L⁻¹), Tris-HCl (50 mmol·L⁻¹), EDTA (1 mmol·L⁻¹), sodium deoxycholate (0.5%), Na₄P₂O₇⋅10H₂O (40 mmol·L⁻¹), NaF (50 mmol·L⁻¹), Na₃VO₄ (1 mmol·L⁻¹), PMSF (2 mmol·L⁻¹) and cOmplete^TM Protease Inhibitor Cocktail (Roche Bioscience). The extracted proteins underwent SDS–PAGE and were transferred to PVDF membranes (pore size: 0.45 μm, Merck), followed by immunoblotting using the following antibodies: PLCγ1 (1/1 000, CST), phospho–PLCγ1 S1248 (1/1 000, CST), and β-actin (1/5 000, Merck). The blots were visualized using the following reagents: horseradish peroxidase (HRP)–linked anti–mouse IgG, HRP–linked anti–rabbit IgG (1/5 000, GE Healthcare), and a luminol reagent (Nacalai Tesque). Densitometric analysis of the bands was conducted using Photoshop 2020 (Adobe).