Anti ha hrp antibody

The plasmid DNA p35S:CCS52A2-FLAG was transformed into protoplasts with or without p35S:FBS1-HA and then incubated at 20°C-23°C for 5 h under light conditions. After that, samples were treated with cycloheximide (CHX; Sigma, USA) with or without the MG132 (Sigma) and incubated for 3 h additionally. Before the treatment of the CHX and MG132, some samples were harvested (0 h samples) using centrifuged at 1,000 × g for 1 min. After 3 h of additional incubation, all samples were harvested and proteins were extracted from the harvested protoplast by boiling with protein sample buffer (60 mM Tris-Cl, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1% Coomassie bromophenol blue).
The prepared protein samples were separated by SDS-PAGE using 10% acrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane containing protein samples was incubated with 1× PBST (Phosphate-buffered saline with 0.1% (v/v) Tween 20) solution containing 6% (w/v) skim milk for 30 min. The membrane was incubated with an anti-HA-HRP antibody (1:1,000 dilution; Roche) and anti-FLAG-HRP (1:1,000 dilution; Sigma) at room temperature (RT) for 1 h 30 min. After incubation, membranes were washed three times with 1× PBST, the chemiluminescence images were developed using ECL reagents (Sigma) and ChemiDocTM XRS+ imaging system (Bio-Rad Laboratories, USA).