Recombinant Anti-BACE1 antibody [EPR3956] (ab108394)

False colour image of Western blot: Anti-BACE1 antibody [EPR3956] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab108394 was shown to bind specifically to BACE1. A band was observed at 60/70 kDa in wild-type SH-SY5Y cell lysates with no signal observed at this size in Bace1 knockout cell line ab280078 (knockout cell lysate ab280137).

To generate this image, wild-type and Bace1 knockout SH-SY5Y cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: BACE1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: SHSY5Y whole cell lysate (20 µg)
Lane 4: Human brain whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab108394 observed at 70 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab108394 was shown to recognize BACE1 in wild type cells as signal was lost at the expected MW in BACE1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and BACE1 knockout samples were subjected to SDS-PAGE. Ab108394 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Blocking and dilution buffer: 5% NFDM/TBST.

Blocking and dilution buffer: 5% NFDM/TBST.

ab108394 (purified) at 1/40 immunoprecipitating BACE1 in human fetal brain whole tissue lysate.

Lane 1 (input): human fetal brain whole tissue lysate (10µg)

Lane 2 (+): ab108394 + human fetal brain whole tissue lysate (10µg).

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab108394 in human fetal brain whole tissue lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1500 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.