重组Anti-Smad1抗体[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.

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.

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).