Recombinant Anti-BACE1 antibody [EPR19523] - BSA and Azide free (ab238937)

False colour image of Western blot: Anti-BACE1 antibody [EPR19523] 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, ab183612 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.

This data was developed using the same antibody clone in a different buffer formulation (ab183612).

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)

Lanes 1 - 3: Merged signal (red and green). Green - ab183612 observed at 68 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab183612 was shown to specifically react with BACE1 in wild-type HAP1 cells as signal was lost in BACE1 knockout cells. Wild-type and BACE1 knockout samples were subjected to SDS-PAGE. ab183612 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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 1/10000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labelling BACE1 with ab183612 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. The sample was counterstained with hematoxylin. Antigen retrieval was performed using Tris/EDTA buffer; pH 9.0.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Binding in rat was weak under our experimental conditions and requires further optimization.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).

Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue labelling BACE1 with ab183612 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. The sample was counterstained with hematoxylin. Antigen retrieval was performed using Tris/EDTA buffer; pH 9.0.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Binding in rat was weak under our experimental conditions and requires further optimization.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling BACE1 with ab183612 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Negative staining on mouse liver. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse hippocampus tissue labeling BACE1 with ab183612 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on mouse Hilar region of the dentate gyrus is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling BACE1 with ab183612 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on neurons of the mouse cerebrum is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling BACE1 with ab183612 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on some neurons of the rat cerebrum is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Binding in rat was weak under our experimental conditions and requires further optimization.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen mouse hippocampus tissue labeling BACE1 with ab183612 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).  The result showed mainly cytoplasmic staining on mouse hippocampus. The nuclear counterstain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).

BACE1 was immunoprecipitated from 1mg of rat hippocampus whole cell lysate with ab183612 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab183612 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Rat hippocampus whole cell lysate,  10µg (Input).

Lane 2: ab183612 IP in Rat hippocampus whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab183612 in rat hippocampus whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 5 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).

BACE1 was immunoprecipitated from 1mg of mouse hippocampus whole cell lysate with ab183612 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab183612 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: Mouse hippocampus whole cell lysate, 10µg (Input).

Lane 2: ab183612 IP in mouse hippocampus whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab183612 in Mouse hippocampus whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab183612).