Anti rabbit igg conjugated with horseradish peroxidase

Protein samples were diluted with Laemmli buffer (Bio‐Rad Laboratories) to 1 μg/μL and heat‐denatured for 4 min at 96°C. Samples (10 μg/lane) were loaded on a 12.5% polyacrylamide gel. The separated proteins were then transferred from the gel to a nitrocellulose membrane (GE Healthcare Inc., Piscataway, NJ) for 1 h at room temperature, and blocked for 1 h at room temperature in 3% blocking reagent (GE Healthcare Inc.) in tris buffered saline (TBS) buffer with 0.1% Tween 20 (TBS‐T). Membranes were incubated overnight at 4°C with rabbit monoclonal anti‐Akt (1:1000) or anti‐phospho‐Akt (pAkt) (ser473) (1:2000) antibodies (both from Cell Signaling Technology Inc, Danvers, MA) in TBS buffer. The subsequent incubation with secondary antibody (anti‐rabbit IgG) conjugated with horseradish peroxidase (1:15000) (GE Healthcare Inc.) was performed at room temperature for 1 h. The signals were developed using enhanced chemiluminescence (ECL) prime reagent (GE Healthcare Inc.) and imaged (Lumi‐Imager F1 LumiImager, Roche Mannheim Boehringer, Mannheim, Germany). The band intensities were determined using ImageJ Software (NIH).

Rabbit polyclonal anti-MuLVp30^CA antiserum [47 (link)] and anti-KoRV CA [14 (link)] antiserum were described previously. For detection of epitope tags, mouse and rabbit anti-Flag antibodies (Cell Signaling), anti-V5 (Invitrogen) and anti-GFP (Biovision) were used. Beta-Tubulin levels were used to assess sample loading in the gels and were detected by rabbit anti-beta-Tubulin (Cell Signaling). For western blots, we used anti-mouse IgG conjugated with horseradish peroxidase (Thermo Scientific) and anti-rabbit IgG conjugated with horseradish peroxidase (GE Healthcare).