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.

All lanes : Anti-BACE1 antibody [EPR19523] (ab183612) at 1/1000 dilution

Lane 1 : Mouse hippocampus lysate
Lane 2 : Rat brain lysate
Lane 3 : Rat hippocampus lysate
Lane 4 : Mouse ovary lysate
Lane 5 : Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate
Lane 6 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 7 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 56 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 1 minute; Lane 3,4,5,6,7 and 8: 3 minutes.

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.

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

Anti-BACE1 antibody [EPR19523] (ab183612) at 1/1000 dilution + Mouse brain lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 56 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

An additional band was observed at 70 kD. The expression profile is consistent with what has been described in the literature (PMID: 22741101).

Blocking/Dilution buffer: 5% NFDM/TBST.

 

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.

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.

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

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.

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

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.

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.

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.

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.

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.