Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Calpain small subunit 1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: A431 whole cell lysate (20 µg)
Lane 4: HEK293 whole cell lysate (20 µg)

 

Lanes 1 - 4: Merged signal (red and green). Green - ab92333 observed at 28 kDa. Red - loading control, ab8245, observed at 37 kDa.

 

ab92333 was shown to recognize Calpain small subunit 1 when Calpain small subunit 1 knockout samples were used, along with additional cross-reactive bands. Wild-type and Calpain small subunit 1 knockout samples were subjected to SDS-PAGE. Ab92333 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Calpain small subunit 1 antibody [EPR3324] (ab92333) at 1/2000 dilution

Lane 1 : T47D
cell lysate
Lane 2 : fetal brain lysate
Lane 3 : fetal spleen lysate
Lane 4 : 293T
cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 28 kDa
Observed band size: 28 kDa

Immunohistochemistry staining of Calpain small subunit 1 in formalin-fixed, paraffin-embedded Human kidney tissue using 1/100 ab92333.

Heat mediated antigen retrieval was performed via the pressure cooker method before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with ab92333 (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 (ab92333, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.