Anti-Beclin 1 抗体 [EPR19662]

• IP

Supplier Data

Immunoprecipitation - Anti-Beclin 1 antibody [EPR19662] (AB207612)

Beclin 1 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab207612 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab207612 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

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

Lane 2 : ab207612 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207612 in HeLa whole cell lysate.

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-Beclin 1 antibody [EPR19662] (ab207612)

Predicted band size: 52 kDa

false

• IP

Collaborator

Immunoprecipitation - Anti-Beclin 1 antibody [EPR19662] (AB207612)

Immunoprecipitation of BECN1 in HeLa cells. Lysates were prepared and immunoprecipitation was performed using 2 μg of ab207612 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

This data was 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.

false

• WB

Supplier Data

Western blot - Anti-Beclin 1 antibody [EPR19662] (AB207612)

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

In Western blot, ab325242 was shown to react with Beclin-1 (phospho S90) in wild-type HeLa serum starved overnight then treated with 100nM Calyculin A for 30 minutes lysate(lane2) with loss of signal observed in BECN1 knockout HeLa serum starved overnight then treated with 100nM Calyculin A for 30 minutes lysate(lane3).

The identity of the higher MW bands at approximately 70KDa and 250KDa are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Beclin 1 antibody [EPR19662] (ab207612) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Beclin-1 (phospho S90) antibody [EPR26903-61R] (<a href='/products/primary-antibodies/beclin-1-phospho-s90-antibody-epr26903-61r-ab325242'>ab325242</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 80 µg

Lane 2:

HeLa serum starved overnight then treated with 100nM Calyculin A for 30 minutes, whole cell lysate (untreated membrane) at 80 µg

Lane 3:

BECN1 knock-out HeLa serum starved overnight then treated with 100nM Calyculin A for 30 minutes, whole cell lysate (untreated membrane) at 80 µg

Lane 4:

HeLa serum starved overnight then treated with 100nM Calyculin A for 30 minutes, whole cell lysate (phosphatase treated membrane) at 80 µg

Lane 5:

BECN1 knock-out HeLa serum starved overnight then treated with 100nM Calyculin A for 30 minutes, whole cell lysate (phosphatase treated membrane) at 80 µg

Secondary

All lanes:

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

Observed band size: 60 kDa,36 kDa,52 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Beclin 1 antibody [EPR19662] (AB207612)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Beclin 1 antibody [EPR19662] (ab207612) at 1/2000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-Beclin 1 antibody [EPR19662] (AB207612)

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

The identity of the higher MW band at approximately 70KDa is unknown

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Beclin 1 antibody [EPR19662] (ab207612) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Beclin-1 (phospho S90) antibody [EPR26903-61R] (<a href='/products/primary-antibodies/beclin-1-phospho-s90-antibody-epr26903-61r-ab325242'>ab325242</a>) at 1/1000 dilution

Lane 1:

Untreated HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 80 µg

Lane 2:

HEK-293 treated with 100nM Calyculin A for 30 minutes whole cell lysate at 80 µg

Lane 3:

Untreated MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 80 µg

Lane 4:

MCF7 treated with 100nM Calyculin A for 30 minutes whole cell lysate at 80 µg

Secondary

All lanes:

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

Observed band size: 60 kDa,36 kDa,52 kDa

false

Exposure time: 180s

• WB

Collaborator

Western blot - Anti-Beclin 1 antibody [EPR19662] (AB207612)

ab207612 was shown to react with BECN1 in wild-type HeLa cells in Western blot with loss of signal observed in BECN1 knockout cell line ab262511. Wild-type HeLa and BECN1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab207612 overnight at 4 °C at a 1/2000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was 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-Beclin 1 antibody [EPR19662] (ab207612) at 1/2000 dilution

Lane 1:

Wild-type HeLa lysate at 30 µg

Lane 2:

BECN1 knock-out HeLa lysate at 30 µg

Lane 2:

Western blot - Human BECN1 (Beclin 1) knockout HeLa cell line (<a href='/products/cell-lines/human-becn1-beclin-1-knockout-hela-cell-line-ab262511'>ab262511</a>) at 30 µg

false

• WB

Lab

Western blot - Anti-Beclin 1 antibody [EPR19662] (AB207612)

Western blot : Anti-BECN1 antibody [EPR19662] (ab207612) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab207612 was shown to bind specifically to BECN1. A band was observed at 57 kDa in wild-type HeLa cell lysates with no signal observed at this size in BECN1 knockout cell line. To generate this image, wild-type and BECN1 knockout HeLa 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.

All lanes:

Western blot - Anti-Beclin 1 antibody [EPR19662] (ab207612) at 1/2000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

BECN1 knockout HeLa cell lysate at 20 µg

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

HepG2 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

false

• WB

Supplier Data

Western blot - Anti-Beclin 1 antibody [EPR19662] (AB207612)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1-4 : 30 seconds; Lane 5 : 15 seconds; Lane 6 : 15 seconds.

All lanes:

Western blot - Anti-Beclin 1 antibody [EPR19662] (ab207612) at 1/5000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 10 µg

Lane 3:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 5:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

Lane 6:

Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

• WB

Supplier Data

Western blot - Anti-Beclin 1 antibody [EPR19662] (AB207612)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/6 : 5 seconds; Lane 2/3 : 15 seconds; Lane 4 : 3 seconds; Lane 5 : 10 seconds.

All lanes:

Western blot - Anti-Beclin 1 antibody [EPR19662] (ab207612) at 1/2000 dilution

Lane 1:

Rat brain lysate at 10 µg

Lane 2:

Rat kidney lysate at 10 µg

Lane 3:

Rat spleen lysate at 10 µg

Lane 4:

Mouse brain lysate at 10 µg

Lane 5:

Mouse heart lysate at 10 µg

Lane 6:

Mouse spleen lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

• WB

Supplier Data

Western blot - Anti-Beclin 1 antibody [EPR19662] (AB207612)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1-4 : 5 seconds; Lane 5 : 10 seconds; Lane 6 : 30 seconds.

Lanes 1 - 4:

Western blot - Anti-Beclin 1 antibody [EPR19662] (ab207612) at 1/2000 dilution

Lanes 5 - 6:

Western blot - Anti-Beclin 1 antibody [EPR19662] (ab207612) at 1/5000 dilution

Lane 1:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 2:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Lane 5:

A20 (Mouse reticulum sarcoma cell line) whole cell lysate at 10 µg

Lane 6:

C2C12 (Mouse myoblast cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase at 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

• WB

Supplier Data

Western blot - Anti-Beclin 1 antibody [EPR19662] (AB207612)

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

CaMKII has been shown to directly phosphorylate Beclin 1 at Ser90 (PMID : 29079782)

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

In Western blot, Anti-Beclin 1 antibody [EPR19662] (ab207612) staining at 1/1000 dilution.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Beclin-1 (phospho S90) antibody [EPR26903-61R] (<a href='/products/primary-antibodies/beclin-1-phospho-s90-antibody-epr26903-61r-ab325242'>ab325242</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 2:

293T transfected with human BECN1 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 3:

293T transfected with human BECN1 expression vector containing a myc-His-tag® transfected with a human wild-type CaMKII beta expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Lane 4:

293T transfected with human BECN1(S90A mutant) expression vector containing a myc-His-tag® whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 60 kDa,80 kDa,52 kDa,36 kDa

false

Exposure time: 8s