Anti-beta Amyloid antibody [2E9]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Amyloid antibody [2E9] (AB252816)

Immunohistochemical analysis of paraffin-embedded Alzheimer's Disease cerebrum tissue labeling beta Amyloid with ab252816
at 0.583μg/ml, followed by ready to use secondary ab214882. Positive staining on Alzheimer's Disease cerebrum tissue is observed (PMID : 1640495). The section was incubated with ab252816 for 30 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, followed by ready to use secondary. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-beta Amyloid antibody [2E9] (AB252816)

ab252816 was shown to react with APP in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a APP knockout cell line. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab252816 overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 µg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• IP

Supplier Data

Immunoprecipitation - Anti-beta Amyloid antibody [2E9] (AB252816)

Beta Amyloid was immunoprecipitated from 1mg of HeLa (human cervix adenocarcinoma epithelial cell line) whole cell lysate with ab252816 at 19.443. Western blot was performed from the immunoprecipitate using ab252816 at 1/1000 dilution. Goat-anti-Rat IgG for IP (HRP) (ab205720) was used at 1/1000 dilution.

Lane 1 : HeLa whole cell lysate 10μg (Input).

Lane 2 : ab252816 IP in HeLa whole cell lysate.

Lane 3 : Rat monoclonal IgG instead of ab252816 in HeLa whole cell lysate.

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

Exposure time : 5 secs.

All lanes:

Immunoprecipitation - Anti-beta Amyloid antibody [2E9] (ab252816)

Predicted band size: 87 kDa

false

• WB

Lab

Western blot - Anti-beta Amyloid antibody [2E9] (AB252816)

ab252816 was shown to react with APP in wild-type HAP1 cells in Western blot with loss of signal observed in a APP knockout cell line. Wild-type HAP1 and APP knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab252816 overnight at 4 °C at a 1/500 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-beta Amyloid antibody [2E9] (ab252816) at 1/500 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

APP Knockout HAP1 lysate at 20 µg

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-beta Amyloid antibody [2E9] (AB252816)

Western blot : Anti-APP antibody [2E9] (ab252816) staining at 0.583 ug/ml, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab252816 was shown to bind specifically to APP. A band was observed at 80-130 kDa in wild-type HAP1 and HEK-293T cell lysates with no signal observed at this size in both APP knockout cell lines. To generate this image, wild-type and APP knockout HAP1 and HEK-293T 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-Rat IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

Lanes 1 - 7:

Western blot - Anti-beta Amyloid antibody [2E9] (ab252816) at 0.583 µg/mL

Lanes 1 - 7:

Western blot - Anti-beta Amyloid antibody [2E9] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/beta-amyloid-antibody-2e9-bsa-and-azide-free-ab255772'>ab255772</a>)

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

APP knockout HAP1 cell lysate at 20 µg

Lane 3:

Wild-type HEK-293T cell lysate at 20 µg

Lane 4:

APP knockout HEK-293T cell lysate at 20 µg

Lane 5:

Human Hippocampus cell lysate at 20 µg

Lane 6:

U-87 MG cell lysate at 20 µg

Lane 7:

K562 cell lysate at 20 µg

Observed band size: 80-130 kDa

false

• WB

Supplier Data

Western blot - Anti-beta Amyloid antibody [2E9] (AB252816)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lanes 1-3 : 37 secs; Lanes 4-5 : 5 secs.

All lanes:

Western blot - Anti-beta Amyloid antibody [2E9] (ab252816) at 0.583 µg/mL

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate at 10 µg

Lane 2:

HEK-293 (human embryonic kidney epithelial cell), whole cell lysate at 10 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 10 µg

Lane 4:

Human brain tissue lysate at 10 µg

Lane 5:

Human hippocampus tissue lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/5000 dilution

Predicted band size: 87 kDa

false