Anti-Apolipoprotein E antibody (ab20874)

Western blot - Apolipoprotein E antibody (ab20874)

Anti-Apolipoprotein E antibody (ab20874) + 20ug rat hippocampal lysate

Secondary
anti-rabit HRP-labeled secondary antibody

Predicted band size : 36 kDa

Rabbit polyclonal to Apolipoprotein E (ab20874) in western blot analysis of proteins isolated from 100mg of rat brain hippocampaus. Tissue was homogenized in a 5V of gentle lysis buffer (10mM HEPES, 1mM EGTA, 5mM MgCl2, 142,5mM KCl, 0,25% NP-40, with Roche complete Protease Inhibitor Cocktail Tablets) and centrifuged 20 mins/14000rpm/4^oC. Supernatants reserved and protein concentrations were assigned by Bradford method. Proteins were resolved onto 10% SDS-PAGE and blotted on nitrocellulose membranes (Amersham- Hybond C). The membranes were incubated with anti-ApoE (ab20874 at 1/400) for 2h at rm temp in 0.05% TBST (150 mM NaCl, 50 mM Tris, pH 7.0, and 0.05% Tween 20) with 3% milk. Following incubation with the anti-rabbit HRP-labeled secondary antibody (1/10000 in 0.05% TBST, 1h rm temp), blots were developed using the enhanced chemiluminescent substrate (ECL, Amersham Bioscience).

S Kanazir, Inst Biol Res, Yugoslavia

Immunohistochemistry (Frozen sections) - Apolipoprotein E antibody (ab20874)

ab20874 staining Apolipoprotein E in rat brain tissue by Immunohistochemistry (Frozen sections). After fixation in 4% PFA in PBS (pH 7.4), brains were cryoprotected in 30% sucrose in PBS at +4ºC, frozen, and thereafter kept at -80ºC until used. For the immunostaining, brains were cryosectioned in 30 µm slices, collected in PBS (pH 7.4), and processed in a free-floating system. Slices were first incubated in the blocking solution (BS) containing 2% bovine serum albumin, 2% goat serum, and 0.1% Triton X-100 in PBS (pH 7.4) for 1 hour at room temperature, and thereafter with the primary antibody for 24–48 hours at +4ºC in BS at a 1/1000 dilution. After washing in PBS, slices were incubated with the biotin-conjugated secondary antibody (1/4000) in BS, rinsed with PBS, and incubated with the avidin-peroxidase conjugate in BS for 1 hour at room temperature. The staining was detected using 3,3-diaminobenzidinetetrahydrochloride as a chromogen.

Image from Laurén HB et al, PLoS One. 2010 May 20;5(5):e10733, Fig 5.