anti-rabbit HRP-conjugated antibodies (DakoCytomation, P0448, lot 00055814)
Primary mouse microglia are grown in DMEM (Invitrogen), supplemented with 10% foetal bovine serum (Gibco), 10% horse serum and penicillin and streptomycin (Gibco), at 37°C and 5% CO2 in a 12-well dish.
Lyse cells in 25µl per well cold lysis buffer (10mM Tris-HCl pH 8.0, 150mM NaCl, 1% Nonidet-P40, 1mM EDTA, 10mM NaF, 1mM Na3VO4, protease inhibitors (Sigma)).
Determine total protein content of the lysates using Bradford assay (Bio-Rad, 500-0006, lot 111832).
Denature 5µg, 10µg and 30µg total protein for 5min at 95°C in 5µl, 10µl and 20µl Laemmli SDS sample buffer and subsequently separate them on a denaturing self-made 10% SDS-PAGE for 1h at 140V.
Transfer proteins onto PVDF membrane (Pall Life Sciences, 75696G, lot TO3225) with a semi-dry Western blotting system for 50min at 60mA.
Block the membrane with TBST (TBS, 0.1% Tween) containing 5% milk for 1h at RT.
Wash membrane 3x 5min with TBST.
Incubation with primary rabbit anti-ARHGEF4 antibody (antibodies-online, ABIN2787993, lot QC30159-41213) diluted 1:500 in TBST containing 2% BSA ON at 4°C.
Wash membrane 5x 5min with TBST.
Incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, P0448, lot 00055814) diluted 1:2000 in TBST containing 5% milk for 1h at RT.
Wash membrane 5x 5min with TBST and 3x 5min with TBS.
Reveal protein bands using ECL Plus Western Blotting Detection Reagent (GE Healthcare, RPN2132, lot 4645641) with Curix RP2 Plus medical X-ray films (AGFA Health Care, ENKMV, lot 79040074) on an AGFA Health Care Curix60-developer.
The ARHGEF4 antibody ABIN2787993 reveals multiple bands as is expected for murine Arhgef4.