Polyclonal rabbit anti mouse immunoglobulins hrp

Antibodies used for flow cytometry studies included an SSPE serum (from a 1978 Belfast case), anti-MV-H mAbs 55 (SLAM-binding site) [19 (link)], BH129 (anti-NE epitope) [20 (link)], BH216 (anti-Noose epitope) [21 (link)], and anti-MV-F mAb Y503 (a gift from D. Gerlier, Inserm U758). Secondary antibodies were rabbit anti-human IgG and goat anti-mouse IgG (both from Millipore). For the western blot an anti-MV-H mAb BH195 [20 (link)] and an anti-MV-F mAb (a gift from Christian Buchholz, Paul Ehrlich Institute, Langen, Germany) were used together with secondary antibodies, polyclonal rabbit anti-mouse immunoglobulins/HRP, and polyclonal goat anti-rabbit immunoglobulins/HRP, respectively (Dako). Antibodies used for the confocal microscope studies were anti-MV-H mAb BH129, anti-MV-F mAb Y503, and anti-MV-M mAb 8910 (Millipore).

Equal amounts of LDs (normalized to OD₆₀₀) were mixed with sample buffer, were loaded to and separated in 15% SDS-PAGE gels and then blotted on protran BA85 nitrocellulose membranes (Schleicher & Schuell BioScience GmbH, Dassel, Germany) (250 mA, 90 min at RT). After blocking the membranes with MTBS-T (25 mM TRIS pH 7.6, 137 mM NaCl, 0.1% TWEEN 20, 5% nonfat milk powder) for 90 min at RT and washing for 30 min with TBS-T the primary-antibodies diluted in MTBS-T were added and incubated overnight at 4 °C under constant shaking. The antibodies were diluted as follows: GFP-antibody (B-2) HRP (sc-9996 HRP; Santa Cruz; 1 : 1000); FLAG M2 antibody (F3165; Sigma-Aldrich; 1 : 500); BAX-antibody (D2E11) (5023; Cell Signal Technologies; 1 : 1000); BCL-XL-antibody (54H6) (2764; Cell Signal Technologies; 1 : 1000). After three washing steps with TBS-T (3×10 min at RT) the membranes were incubated with the secondary-antibody, diluted in MTBS-T (5% w/v milk powder in TBS-T) (Polyclonal rabbit anti-mouse immunoglobulins/HRP; P0161; Dako; 1 : 25000). Detection was carried out with the Pierce (Thermo Fisher Scientific, Waltham, MA, USA) ECL western blotting substrate according the manufacturerś instructions.