Anti-Smad1 antibody [EP565Y] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Smad1 antibody [EP565Y] - BSA and Azide free (AB172642)

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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33902).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Smad1 antibody [EP565Y] - BSA and Azide free (AB172642)

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). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33902).

• WB

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Western blot - Anti-Smad1 antibody [EP565Y] - BSA and Azide free (AB172642)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33902).

All lanes:

Western blot - Anti-Smad1 antibody [EP565Y] (<a href='/en-us/products/primary-antibodies/smad1-antibody-ep565y-ab33902'>ab33902</a>) 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='/en-us/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] - BSA and Azide free (AB172642)

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] (<a href='/en-us/products/primary-antibodies/smad1-antibody-ep565y-ab33902'>ab33902</a>) 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='/en-us/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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