Anti-ALKBH1 antibody [EPR19215]

• IP

Supplier Data

Immunoprecipitation - Anti-ALKBH1 antibody [EPR19215] (AB195376)

ALKBH1 was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate with ab195376 at 1/30 dilution.

Western blot was performed from the immunoprecipitate using ab195376 at 1/1000 dilution.

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

Lane 1 : K562 whole cell lysate 10μg (Input).

Lane 2 : ab195376 IP in K562 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab195376 in K562 whole cell lysate.

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

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-ALKBH1 antibody [EPR19215] (ab195376)

Predicted band size: 43 kDa

false

• WB

Supplier Data

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 dilution

Lane 1:

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

Lane 2:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 10 µg

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 10 µg

Lane 4:

A431 (Human epidermoid carcinoma cell line) whole cell lysate at 10 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 1/100000 dilution

Predicted band size: 43 kDa

Observed band size: 44 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 dilution

Lane 1:

Human fetal kidney lysate at 10 µg

Lane 2:

Human colon lysate at 10 µg

Lane 3:

Human fetal liver 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: 43 kDa

Observed band size: 44 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Lanes 1 - 3 : Merged signal (red and green). Green - ab195376 observed at 43 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab195376 was shown to specifically react with in wild-type HAP1 cells as signal was lost in ALKBH1 knockout cells. Wild-type and ALKBH1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab195376 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

ALKBH1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

A549 whole cell lysate at 20 µg

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 dilution

Lane 1:

A549 (Human lung carcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

A549 (Human lung carcinoma epithelial cell) transfected with siRNA specifically targeting ALKBH1 whole cell lysate at 20 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

false

Exposure time: 120s

• WB

Supplier Data

Western blot - Anti-ALKBH1 antibody [EPR19215] (AB195376)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1-4 : 30 seconds Lane 5-7 : 3 minutes.

All lanes:

Western blot - Anti-ALKBH1 antibody [EPR19215] (ab195376) at 1/1000 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:

Mouse brain lysate at 10 µg

Lane 6:

Mouse spleen lysate at 10 µg

Lane 7:

Rat spleen lysate at 10 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 1/100000 dilution

Predicted band size: 43 kDa

Observed band size: 44 kDa

false