Anti-Smad1 antibody [EP565Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Smad1 antibody [EP565Y] (AB33902)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Smad1 with Purified ab33902 at 1/100 dilution (10 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Smad1 antibody [EP565Y] (AB33902)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Smad1 with Purified ab33902 at 1/100 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).

• WB

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Western blot - Anti-Smad1 antibody [EP565Y] (AB33902)

Blocking/Diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Smad1 antibody [EP565Y] (ab33902) at 1/1000 dilution

Lane 1:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 20 µg

Lane 2:

A-673 (Human muscle Ewing's Sarcoma) whole cell lysates at 20 µg

Lane 3:

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

Lane 4:

C2C12 (Mouse myoblasts myoblast) whole cell lysates at 20 µg

Lane 5:

Mouse brain lysates at 20 µg

Lane 6:

Mouse skeletal muscle lysates at 20 µg

Lane 7:

Mouse stomach lysates at 20 µg

Secondary

All lanes:

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

Predicted band size: 52 kDa

Observed band size: 58 kDa

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• WB

Lab

Western blot - Anti-Smad1 antibody [EP565Y] (AB33902)

Western blot : Anti-SMAD1 antibody [EP565Y] (ab33902) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab33902 was shown to bind specifically to SMAD1. A band was observed at 55-65 kDa in wild-type A549 cell lysates with no signal observed at this size in SMAD1 knockout cell line. To generate this image, wild-type and SMAD1 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 5 % 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-Smad1 antibody [EP565Y] (ab33902) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

SMAD1 knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type HeLa ab255929 cell lysate at 20 µg

Lane 4:

SMAD1 knockout HeLa <a href='/ko/products/cell-lines/human-smad1-knockout-hela-cell-line-ab265400'>ab265400</a> 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

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