Hrp conjugated anti mouse igg secondary antibody

Cells were collected and dissolved in sample buffer (final concentrations: 0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.005% bromophenol blue). Western blotting was performed as previously described [50 (link)]. Briefly, after denaturation by heating at 95 degree for 10 min, 10 μg of each sample was separated by 4–20% SDS-PAGE (Bio-Rad) along with a protein standard ladder (Nippon Genetics). The samples were then transferred to PVDF membranes. The membranes were cut and blocked in TBS with 0.1% Tween 20 (TBS-T) and 5% skim milk for 1 h at room temperature, then incubated with primary monoclonal antibodies against Exo1 (1:100, Thermo Scientific, Ab-4, clone 266) and β-Actin (1:1000, Santa Cruz Biotechnology, sc-47778) in TBS-T containing 1% skim milk at 4° overnight. Next, the membranes were washed, incubated with HRP-conjugated anti-mouse IgG secondary antibody (1:5000, Jackson ImmunoResearch Laboratories, 715-035-151) at room temperature for 1 h, washed, and visualized with Luminata Forte Western HRP substrate (Millipore). Gel images were taken every 1 min for 20 min using Image Lab software (Bio-Rad). Band intensities of gel images within linear signal range were quantified using Image Lab software and normalized with band intensities of β-Actin.

Western blot analyses were used to determine whether PAI-1 vaccine-elicited antibodies recognize and bind to recombinant full-length PAI-1 protein (A8111, Sigma-Aldrich Japan). KLH, PAI-1 partial peptide conjugated with BSA, and full-length PAI protein underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis and were blotted onto polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany). The blots were incubated with serum samples from mice immunized with either the vaccine or vehicle, followed by incubation with HRP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The chemiluminescence signal for these blots were visualized by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and detected with an ImageQuant LAS 4000 mini chemiluminescence imager (GE Healthcare Bio-Sciences, Tokyo, Japan).