Anti-c-Myb antibody [EPR27202-86]

• IP

Lab

Immunoprecipitation - Anti-c-Myb antibody [EPR27202-86] (AB324970)

c-Myb was immunoprecipitated from 0.35 mg MOLT-4 (human lymphoblastic leukemia T lymphoblast) fresh whole cell lysate with ab324970 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab324970 at 1/324970 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : MOLT-4 (human lymphoblastic leukemia T lymphoblast) fresh whole cell lysate
Lane 2 : ab324970 IP in MOLT-4 (human lymphoblastic leukemia T lymphoblast) fresh whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab324970 in MOLT-4 whole cell lysate

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

Exposure time : 180 seconds.

All lanes:

Immunoprecipitation - Anti-c-Myb antibody [EPR27202-86] (ab324970) at 1/1000 dilution

Lane 1:

MOLT-4 (human lymphoblastic leukemia T lymphoblast) fresh whole cell lysate at 10 µg

Lane 2:

MOLT-4 (human lymphoblastic leukemia T lymphoblast) fresh whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab324970 in MOLT-4 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 75 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-c-Myb antibody [EPR27202-86] (AB324970)

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

Negative control : COLO 201.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-c-Myb antibody [EPR27202-86] (ab324970) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 80 µg

Lane 2:

COLO 201 (human colon adenocarcinoma tumor ascitic fibroblast-like cell) whole cell lysate at 80 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

Observed band size: 75 kDa,36 kDa

false

Exposure time: 70s

• WB

Lab

Western blot - Anti-c-Myb antibody [EPR27202-86] (AB324970)

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

Performed under reducing conditions.

In Western blot, ab324970 was shown to bind specifically to c-Myb. Target of interest was observed at 75 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in MYB knockout cell line (lane 2).

This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-c-Myb antibody [EPR27202-86] (ab324970) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 80 µg

Lane 2:

MYB knockout HAP1 whole cell lysate at 80 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

Observed band size: 75 kDa,36 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-c-Myb antibody [EPR27202-86] (AB324970)

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

Negative control : HeLa.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-c-Myb antibody [EPR27202-86] (ab324970) at 1/1000 dilution

Lane 1:

MOLT-4 (human lymphoblastic leukemia T lymphoblast) fresh whole cell lysate at 80 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) fresh whole cell lysate at 80 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

Observed band size: 75 kDa,36 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-c-Myb antibody [EPR27202-86] (AB324970)

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

Low expression : MEF.

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-c-Myb antibody [EPR27202-86] (ab324970) at 1/1000 dilution

Lane 1:

EL4 (mouse lymphoma T lymphocyte) fresh whole cell lysate at 80 µg

Lane 2:

MEF (mouse embryo fibroblast) fresh whole cell lysate at 80 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

Observed band size: 75 kDa,36 kDa

false

Exposure time: 70s

• WB

Lab

Western blot - Anti-c-Myb antibody [EPR27202-86] (AB324970)

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

This antibody does not cross-react with human b-Myb and a-Myb.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(30-100KDa).

All lanes:

Western blot - Anti-c-Myb antibody [EPR27202-86] (ab324970) at 1/1000 dilution

Lane 1:

His-tagged human c-Myb recombinant fragment at 10 ng

Lane 2:

His-tagged human b-Myb recombinant fragment at 100 ng

Lane 3:

His-tagged human a-Myb recombinant protein at 50 ng

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 1/100000 dilution

Observed band size: 30 kDa,30-100 kDa

false

Exposure time: 15s