Anti mouse igg ap linked

Cells were suspended and then sonicated in RIPA buffer (150 mM NaCl, 50 mM Tris (pH 8.0), 1% Igepal-630 (Sigma-Aldrich), 0.5% sodium deoxycholate, 1 tablet Complete Mini protease inhibitor cocktail (Roche, Basel, Switzerland)) and mixed end-over-end for 1 h at 4 °C. Lysates were cleared by centrifugation, and the total protein was quantified with a Bradford assay kit according to the manufacturer’s instructions (BioRad, Hercules, CA). Equal amounts of protein were mixed with Laemmli buffer and loaded onto 12% gels and separated by SDS-PAGE. Proteins then were transferred to PVDF membranes using a semi-dry method. Membranes were blocked with PBS_Tween (PBS with 0.1% Tween-20 (v/v)) containing 5% dry milk (w/v), incubated in primary antibody (either 1:2,000 mouse HA.11 clone 16B12 (BioLegend, San Diego, CA, #MMS-101R) or 1:6,000 mouse anti-β tubulin (Hybridoma Bank, Iowa City, IA, #E7)), washed with PBS_Tween, incubated in secondary antibody (1:6,000 anti-mouse IgG AP-linked (Promega, Madison, WI) or anti-mouse IgG HRP-linked (Cell Signaling Technology, Beverly, MA)), and washed again with PBS_Tween. Antibody binding was detected using either BCIP/NBT substrate (Promega) or ECL substrate (HyBlot, Denville Scientific, Hollison, MA). For ECL detection, membranes were exposed to photosensitive film (Denville Scientific). All antibodies were diluted in PBS_Tween containing 5% dry milk.