重组Anti-BACE1抗体[EPR3956]

• IP

Lab

Immunoprecipitation - Anti-BACE1 antibody [EPR3956] (AB108394)

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.

All lanes:

Immunoprecipitation - Anti-BACE1 antibody [EPR3956] (ab108394)

Predicted band size: 39 kDa,56 kDa

Observed band size: 42 kDa,70 kDa

false

• WB

Lab

Western blot - Anti-BACE1 antibody [EPR3956] (AB108394)

Western blot : Anti-BACE1 antibody [EPR3956] (ab108394) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab108394 was shown to bind specifically to BACE1. A band was observed at 56 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in BACE1 knockout cell line. To generate this image, wild-type and BACE1 knockout U-87 MG cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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.

All lanes:

Western blot - Anti-BACE1 antibody [EPR3956] (ab108394) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG cell lysate at 20 µg

Lane 2:

Western blot - Human BACE1 knockout U-87 MG cell line (ab306623)

Lane 2:

BACE1 knockout U-87 MG cell lysate at 20 µg

Lane 3:

Wild-type SH-SY5Y cell lysate at 20 µg

Lane 4:

BACE1 knockout SH-SY5Y <a href='/products/unavailable/human-bace1-knockout-sh-sy5y-cell-line-ab280078'>ab280078</a> cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 56 kDa

false

• WB

Lab

Western blot - Anti-BACE1 antibody [EPR3956] (AB108394)

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.

All lanes:

Western blot - Anti-BACE1 antibody [EPR3956] (ab108394)

Predicted band size: 56 kDa

false

• WB

Lab

Western blot - Anti-BACE1 antibody [EPR3956] (AB108394)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-BACE1 antibody [EPR3956] (ab108394) at 1/10000 dilution

All lanes:

Human fetal brain tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 56 kDa

Observed band size: 70 kDa

false

• WB

Lab

Western blot - 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.

Lanes 1 - 3:

Western blot - Anti-BACE1 antibody [EPR3956] (ab108394) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-BACE1 antibody [EPR3956] - BSA and Azide free (<a href='/products/primary-antibodies/bace1-antibody-epr3956-bsa-and-azide-free-ab237595'>ab237595</a>) at 1/1000 dilution

Lane 1:

Wild-type SH-SY5Y cell lysate at 20 µg

Lane 2:

Bace1 knockout SH-SY5Y cell lysate at 20 µg

Lane 2:

Western blot - Human BACE1 knockout SH-SY5Y cell line (<a href='/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 3:

HAP1 cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 70 kDa,60 kDa

false

• WB

Lab

Western blot - Anti-BACE1 antibody [EPR3956] (AB108394)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-BACE1 antibody [EPR3956] (ab108394) at 1/10000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 56 kDa

Observed band size: 70 kDa

false

• WB

Unknown

Western blot - Anti-BACE1 antibody [EPR3956] (AB108394)

All lanes:

Western blot - Anti-BACE1 antibody [EPR3956] (ab108394) at 1/1000 dilution

Lane 1:

Fetal brain lysate at 10 µg

Lane 2:

SH-SY5Y lysate at 10 µg

Lane 3:

Mouse brain lysate at 10 µg

Lane 4:

Rat brain lysate at 10 µg

Predicted band size: 56 kDa

false