Anti-Smad1 antibody [EPR5522] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Smad1 antibody [EPR5522] - BSA and Azide free (AB176885)

This data was developed using ab126761, the same antibody clone in a different buffer formulation.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Smad1 with purified ab126761 at 1/400 dilution (1 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

Lab

Western blot - Anti-Smad1 antibody [EPR5522] - BSA and Azide free (AB176885)

This data was developed using the same antibody clone in a different buffer formulation (ab126761).

Lanes 1- 4 : Merged signal (red and green). Green - ab126761 observed at 52 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab126761 was shown to react with Smad1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265400 (knockout cell lysate ab257686) was used. Wild-type HeLa and SMAD1 knockout HeLa cell lysates were subjected to SDS-PAGE. ab126761 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Smad1 antibody [EPR5522] (<a href='/en-us/products/primary-antibodies/smad1-antibody-epr5522-ab126761'>ab126761</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SMAD1 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-smad1-knockout-hela-cell-lysate-ab257686'>ab257686</a>) at 20 µg

Lane 3:

HT1080 cell lysate at 20 µg

Lane 4:

Huvec cell lysate at 20 µg

Predicted band size: 52 kDa

Observed band size: 52 kDa

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• WB

Lab

Western blot - Anti-Smad1 antibody [EPR5522] - BSA and Azide free (AB176885)

Western blot : Anti-SMAD1 antibody [EPR5522] (ab126761) 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, ab126761 was shown to bind specifically to SMAD1. A band was observed at 52 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 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-Smad1 antibody [EPR5522] (<a href='/en-us/products/primary-antibodies/smad1-antibody-epr5522-ab126761'>ab126761</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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