Western blot - Anti-GCLM antibody [EPR6667] (ab126704)
All lanes : Anti-GCLM antibody [EPR6667] (ab126704) at 1 µg/ml
Lane 1 : Wild-type HAP1 whole cell lysate Lane 2 : GCLM knockout HAP1 whole cell lysate Lane 3 : HeLa whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 31 kDa
Lanes 1 - 3: Merged signal (red and green). Green - ab126704 observed at 31 kDa. Red - loading control, ab130007, observed at 130 kDa.
ab126704 was shown to recognize GCLM in wild-type HAP1 cells as signal was lost at the expected MW in GCLM knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GCLM knockout samples were subjected to SDS-PAGE. Ab126704 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
Overlay histogram showing HeLa cells stained with ab126704 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab126704, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
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