Mouse polyclonal anti actin antibody

Samples of 100 mg ground frozen leaf tissue were used for extraction of total cellular protein using a phenol-based method (Cahoon et al., 1992) with the minor modification that instead of phenyl-methanesulfonyl fluoride, cOmplete Protease Inhibitor Cocktail (Roche) was added. The protein pellets were dissolved in 1% (w/v) SDS and the protein concentration was determined with the BCA Protein Assay kit (Pierce Instruments). For immunoblot analysis, samples of 20 mg protein were separated by 10% (w/v) SDS-PAGE and blotted onto polyvinylidene difluoride membranes (GE Healthcare). Membranes were treated with blocking buffer (13 Tris-buffered saline, 0.1% [v/v] Tween 20, and 1% [w/v] bovine serum albumin) for 1 h and then incubated with a rabbit polyclonal antiglutamyl-tRNA reductase antibody (Agrisera) for another hour. After incubation with antirabbit secondary antibody (Agrisera), immunodetection was performed with the ECL Prime system (GE Healthcare). For actin detection, the membranes were stripped using Thermo Scientific Restore Plus western blot stripping buffer (Fisher Scientific) according to the manufacturer's instructions, followed by treatment with blocking buffer for 1 h, incubation in mouse polyclonal antiactin antibody (Sigma) for 1 h, incubation in antimouse secondary antibody (Sigma), and detection using the ECL Prime system (GE Healthcare).