Anti-Hamartin 抗体 [EP318Y] (ab40872)

Western blot: Anti-TSC1 antibody [EP318Y] (ab40872) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab40872 was shown to bind specifically to TSC1. A band was observed at 130-150 kDa in wild-type MCF7 cell lysates with no signal observed at this size in TSC1 knockout cell line. To generate this image, wild-type and TSC1 knockout MCF7 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.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Hamartin knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Human skeletal muscle tissue lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab40872 observed at 150 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab40872 was shown to recognize Hamartin when Hamartin knockout samples were used, along with additional cross-reactive bands. Wild-type and Hamartin knockout samples were subjected to SDS-PAGE. ab40872 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/10000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

We are not sure about the nature of the extra band. 

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Hamartin with Purified ab40872 at 1/20 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).