Anti-BACE1 antibody [EPR19523] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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

Immunohistochemical analysis of paraffin-embedded human cerebellum tissue* labelling BACE1 with ab183612 at 0.2ug/ml followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human cerebellum.

The section was incubated with ab183612 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0 epitope retrieval solution1) for 20 mins.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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).

• IP

Supplier Data

Immunoprecipitation - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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).

All lanes:

Immunoprecipitation - Anti-BACE1 antibody [EPR19523] (<a href='/en-us/products/primary-antibodies/bace1-antibody-epr19523-ab183612'>ab183612</a>)

Predicted band size: 56 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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).

All lanes:

Immunoprecipitation - Anti-BACE1 antibody [EPR19523] (<a href='/en-us/products/primary-antibodies/bace1-antibody-epr19523-ab183612'>ab183612</a>)

Predicted band size: 56 kDa

false

• WB

Supplier Data

Western blot - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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

Blocking and dilution buffer : 5% NFDM/TBST.

ab181602 was used as a loading control at 1/1000000 dilution.

We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results.

ab263901 could be an alternative for getting stronger signal in testing cell lines.

Lanes 1 - 3:

Western blot - Anti-BACE1 antibody [EPR19523] (<a href='/en-us/products/primary-antibodies/bace1-antibody-epr19523-ab183612'>ab183612</a>) at 1/1000 dilution

Lanes 4 - 6:

Western blot - Anti-BACE1 antibody [EPR22802-233] (<a href='/en-us/products/primary-antibodies/bace1-antibody-epr22802-233-ab263901'>ab263901</a>) at 1/1000 dilution

Lanes 1 and 4:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lanes 2 and 5:

Mouse brain tissue lysate at 20 µg

Lanes 3 and 6:

Neuro-2a (Mouse neuroblastoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 56 kDa

Observed band size: 68 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - 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).

Lanes 1 - 3:

Western blot - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (ab238937) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-BACE1 antibody [EPR19523] (<a href='/en-us/products/primary-antibodies/bace1-antibody-epr19523-ab183612'>ab183612</a>) at 1/1000 dilution

Lane 1:

Wild-type SH-SY5Y cell lysate at 20 µg

Lane 2:

Western blot - Human BACE1 knockout SH-SY5Y cell line (<a href='/en-us/products/unavailable/human-bace1-knockout-sh-sy5y-cell-line-ab280078'>ab280078</a>)

Lane 2:

Western blot - Human BACE1 knockout SH-SY5Y cell lysate (ab280137)

Lane 2:

Bace1 knockout SH-SY5Y cell lysate at 20 µg

Lane 3:

HAP1 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 60 kDa,70 kDa

false

• WB

Supplier Data

Western blot - Anti-BACE1 antibody [EPR19523] - BSA and Azide free (AB238937)

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).

All lanes:

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

Predicted band size: 56 kDa

Observed band size: 68 kDa

false