Goat anti rabbit igg p0448

Western blots were prepared essentially as described in [50 (link)]. Fifteen to thirty μg protein were reduced, denatured and electrophoretically separated on a 12% polyacrylamide gel with a 5.2% polyacrylamide stacking gel on top. Gels were electroblotted onto PVDF Plus transfer membranes (Amersham Hybond-P, GE Healthcare, Buckinghamshire, England) and the membranes were blocked and then incubated with antibodies. Primary antibodies for detecting total-FoxO1 (rabbit monoclonal) (C29H4) [2880] and pFoxO1 S256 (rabbit polyclonal) [9461] were obtained from Cell Signaling Technology (Beverly, CA), MuRF1 (goat polyclonal) [AF5366] was obtained from R&D systems (Abingdon, England) and Ac-FoxO1 (rabbit polyclonal) (FKHR D19) [49437] from Santa Cruz Biotechnology (Santa Cruz, CA). All primary antibodies were used at a dilution of 1/800 – 1/1500. Antibodies were visualized with horseradish peroxidise conjugated secondary immunoglobulin diluted 1/1000 goat anti-rabbit IgG [P0448] and 1/1000-1/10000 rabbit anti-goat IgG [P0449] (Dako, Glostrup, Denmark). The bound immune complexes were detected using the ECL Plus Western blotting detection system and Hyperfilm ECL (Amersham International and Amersham Pharmacia Biotech, Buckinghamshire, England).

The primary and secondary antibodies used for western blot analysis are listed below. The mouse anti-KSHV K-bZIP (sc-69797) was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The primary mouse antibody anti-β-actin (A2228) was purchased from Sigma-Aldrich (Taufkirchen, Germany). For the detection of KSHV LANA, we used a rat monoclonal anti-LANA antibody reported previously [77 (link)]. The HRP-conjugated rabbit anti-rat IgG antibody (3050–05) was purchased from SouthernBiotech (Birmingham, USA), and the HRP-conjugated rabbit anti-mouse IgG (P0447) and goat anti-rabbit IgG (P0448) were purchased from Dako (Santa Clara, USA). The primary mouse antibody anti-ORF26 (clone 2F6B8) was purchased from LSBio (Seattle, USA) and the Cy-3 coupled secondary antibody (Cy3 AffiniPure Donkey Anti-Mouse IgG) from Jackson ImmunoResearch (Ely, Cambridgeshire, United Kingdom).