anti-goat/sheep HRP-conjugated antibodies (DakoCytomation, P0449, lot 00062104)
Primary mouse microglia and human MCF-7 cells 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 plate or in RPMI 1640 (Invitrogen), supplemented with 6% inactivated foetal bovine serum (Gibco), GlutaMax (Gibco), penicillin and streptomycin (Gibco), at 37°C and 5% CO2 in a 10cm dish, respectively.
Lyse microglia in 25µl per well and MCF-7 cells in 500µl per dish cold lysis buffer (10mM Tris-HCl pH 8.0, 150mM NaCl, 1% Nonidet-P40, 1mM EDTA, 10mM NaF, 1mM Na3VO4, containing protease inhibitors (Sigma)).
Determine total protein content of the lysates using Bradford assay (Bio-Rad, 500-0006, lot 111832).
Denature 30µg total protein for 5min at 95°C in in 20µl Laemmli SDS sample buffer and subsequently separate them on a denaturing, freshly cast 7.5% SDS-PAGE for 1h at 140V.
Transfer proteins onto PVDF membrane (Pall Life Sciences, 75696G, lot TO3225) with a wet Western blotting system for 80min at 100V.
Block the membrane with TBST (TBS, 0.1% Tween) containing 5% milk ON at 4°C.
Incubation with primary sheep anti-TIAM1 antibody (antibodies-online, ABIN1982879, lot 2016090702) diluted 1:500 in TBST containing 5% milk for 1h at RT.
Wash membrane 5x 5min with TBST.
Incubation with anti-goat/sheep horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, P0449, lot 00062104) 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 self-made ECL Western Blotting Detection Reagent with Curix RP2 Plus medical X-ray films (Agfa, ENKMV, lot 79040074) on an Agfa- Curix60-developer.
The TIAM1 antibody ABIN1982879 reveals a protein of the expected molecular weight of 177kDa in human MCF-7 lysates as verified with a second anti-TIAM1 antibody. However, note that ABIN1982879 did not recognize Tiam1 in primary mouse microglia extracts.