Anti-Chk1 antibody [E250] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Chk1 antibody [E250] - BSA and Azide free (AB239821)

This data was developed using ab32531, the same antibody clone in a different buffer formulation. Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling Chk1 with purified ab32531 at 1/30 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IP

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Immunoprecipitation - Anti-Chk1 antibody [E250] - BSA and Azide free (AB239821)

This data was developed using ab32531, the same antibody clone in a different buffer formulation.
Purified ab32531 at 1/20 dilution (1μg) immunoprecipitating Chk1 in K-562 whole cell lysate.
Lane 1 (input) : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10μg
Lane 2 (+) : ab32531 + K-562 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32531 in K-562 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 54 kDa

All lanes:

Immunoprecipitation - Anti-Chk1 antibody [E250] (<a href='/en-us/products/primary-antibodies/chk1-antibody-e250-ab32531'>ab32531</a>)

Predicted band size: 54 kDa

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• WB

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Western blot - Anti-Chk1 antibody [E250] - BSA and Azide free (AB239821)

All lanes:

Purfiied at 1/1000 dilution

Lane 1:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

Predicted band size: 54 kDa

Observed band size: 54 kDa

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• WB

Lab

Western blot - Anti-Chk1 antibody [E250] - BSA and Azide free (AB239821)

This data was developed using ab32531, the same antibody clone in a different buffer formulation. Anti-CHEK1 antibody [E250] (ab32531) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32531 was shown to bind specifically to CHEK1. A band was observed at 57 kDa in wild-type A549 cell lysates with a reduction in signal observed at this size in CHEK1 heterozygous knockout cell line. To generate this image, wild-type and CHEK1 heterozygous knockout A549 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-Chk1 antibody [E250] (<a href='/en-us/products/primary-antibodies/chk1-antibody-e250-ab32531'>ab32531</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Western blot - Human CHEK1 heterozygous knockout A549 cell line (<a href='/en-us/products/cell-lines/human-chek1-heterozygous-knockout-a549-cell-line-ab276102'>ab276102</a>)

Lane 2:

CHEK1 knockout A549 cell lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

MOLT-4 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 54 kDa

Observed band size: 57 kDa

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• WB

Unknown

Western blot - Anti-Chk1 antibody [E250] - BSA and Azide free (AB239821)

This data was developed using ab32531, the same antibody clone in a different buffer formulation.

We are unsure how to define the extra bands.

All lanes:

Western blot - Anti-Chk1 antibody [E250] (<a href='/en-us/products/primary-antibodies/chk1-antibody-e250-ab32531'>ab32531</a>) at 1/10000 dilution

Lane 1:

MEF (Mouse embryonic fibroblast (immortalized)) whole cell lysate at 15 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma ) whole cell lysate at 15 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

Predicted band size: 54 kDa

Observed band size: 54 kDa

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