Anti-Smad1 antibody [EPR5522]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Smad1 antibody [EPR5522] (AB126761)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Smad1 antibody [EPR5522] (AB126761)

ab126761, at 1/50 dilution, staining Smad1 in paraffin-embedded Human colon tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-Smad1 antibody [EPR5522] (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] (ab126761) 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='/ko/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

false

• WB

Lab

Western blot - Anti-Smad1 antibody [EPR5522] (AB126761)

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] (ab126761) 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

false

• WB

Unknown

Western blot - Anti-Smad1 antibody [EPR5522] (AB126761)

All lanes:

Western blot - Anti-Smad1 antibody [EPR5522] (ab126761) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

A673 cell lysate at 10 µg

Lane 3:

HepG2 cell lysate at 10 µg

Lane 4:

293T cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit at 1/2000 dilution

Predicted band size: 52 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Smad1 antibody [EPR5522] (AB126761)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.