Anti mouse igg antibodies conjugated to horseradish peroxidase

Whole-cell protein lysates were prepared using RIPA lysis buffer as recently described (46 (link)). Lysates were separated by SDS-PAGE, transferred on a nitrocellulose blotting membrane (0.45 µm), and detected via western blotting as previously described (88 (link)). As primary antibody for HBV Core protein, mouse mAb 8C9-11 (71 (link)), mouse mAb sc-23945 (Santa Cruz), and HBV Capsid rabbit pAb B0586 (DAKO) were used. Cellular protein steady-state levels were determined using polyclonal rabbit Ab raised against PML protein (NB100-59787; Novus Biologicals), and β-actin mouse mAb AC-15 as a control (Sigma-Aldrich, Inc.). mAb3F10 was used for the detection of HA-tagged proteins. To detect proteins by immunoblotting, secondary anti-rabbit IgG and anti-mouse IgG antibodies conjugated to horseradish peroxidase (Jackson/Dianova) were used.
Western blots were processed using Adobe Photoshop CS5 and Adobe Illustrator CS5 software. For relative quantification of protein steady-state levels and comparisons, ImageJ 1.52a (89 (link)) was used.

Samples were prepared with Tris-glycine SDS buffer (Novex, Life Technologies) containing 10% β-ME at a 1:1 ratio (v/v) and heated at 95°C for 5 min. Equal amounts of protein were loaded into 10-well 4–20% Tris-glycine gels (Novex). After transfer, blots were blocked with 5% nonfat dry milk in TBST for 1 h and then incubated with rabbit anti-sera (1:5000), B cell supernatant (1:500), mAbs (26H10, 2E9 and 23A1; 1:500), rabbit polyclonal GFP antibody (1:1000, Thermo Fisher Scientific, A-11122), rabbit polyclonal TDP-43 antibody (1:1000, Proteintech, 12892-1-AP) or mouse monoclonal GAPDH antibody (1:5000, Meridian Bioscience, H86504M) overnight at 4°C with rocking. Membranes were then washed in TBST three times for 10 min each and incubated for 1 h with donkey anti-rabbit or anti-mouse IgG antibodies conjugated to horseradish peroxidase (1:5000, Jackson ImmunoResearch) for 1 h at room temperature. Protein expression was visualized by enhanced chemiluminescence treatment using Western Lightning Plus-ECL (Perkin Elmer).