Anti-Amyloid Precursor Protein antibody [EPR5118-34]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (AB126732)

Overlay histogram showing A431 cells stained with ab126732 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab126732, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (AB126732)

ab126732, at a dilution of 1/100, staining Amyloid Precursor Protein in paraffin-embedded Human fetal brain tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (AB126732)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of HEK-293 whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab126732 (1/1000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab126732 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (ab126732) at 1/1000 dilution

All lanes:

HEK-293 whole cell lysate at 15 µg with NDFM/TBST

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

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Exposure time: 80s

• WB

Lab

Western blot - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (AB126732)

ab126732 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 ab126732 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-Amyloid Precursor Protein antibody [EPR5118-34] (ab126732) 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

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• WB

Lab

Western blot - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (AB126732)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : APP knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HepG2 whole cell lysate (20 μg)
Lane 4 : HeLa whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab126732 observed at 110 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab126732 was shown to specifically react with APP when APP knockout samples were used. Wild-type and APP knockout samples were subjected to SDS-PAGE. ab126732 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 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:

Western blot - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (ab126732)

Predicted band size: 86 kDa

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• WB

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Western blot - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (AB126732)

All lanes:

Western blot - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (ab126732) at 1/1000 dilution

Lane 1:

SH-SY5Y cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

U-87 MG cell lysate at 10 µg

Lane 4:

Neuro 2a cell lysate at 10 µg

Lane 5:

C6 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 86 kDa

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• WB

CiteAb

Western blot - Anti-Amyloid Precursor Protein antibody [EPR5118-34] (AB126732)

Amyloid Precursor Protein western blot using anti-Amyloid Precursor Protein antibody [EPR5118-34] ab126732. Publication image and figure legend from Mendsaikhan, A., Takeuchi, S., et al., 2019, Front Mol Neurosci, PubMed 30670947.

ab126732 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab126732 please see the product overview.

Expression of neuronal disease-associated proteins in FtMt clones. (A) Relative levels of amyloid precursor protein (APP) in different FtMt clones. Results shown represent mean ± SEM of triplicate experiments. Data analyzed by One way ANOVA with Tukey's post-hoc test of significance. Results indicate level of significance. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Relative levels of Tau in different FtMt clones. Results shown represent mean ± SEM of triplicate experiments. Data analyzed by One way ANOVA with Tukey's post-hoc test of significance. Results indicate level of significance. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. (C). Relative levels of α-synuclein (α-syn) in different FtMt clones. Results shown represent mean ± SEM of triplicate experiments. Data analyzed by One way ANOVA with Tukey's post-hoc test of significance. Results indicate level of significance. *p < 0.05. (D) Composite Western blot showing pattern of expression for APP, tau, and α-synuclein. Representative western blots of one experiment showing patterns of expression in untransfected and different FtMt clones. Experiment represents the sequential probing of single membrane for APP, tau, and α-synuclein followed by normalization for β-actin.

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