Anti-Calpain small subunit 1 antibody [EPR3324] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Calpain small subunit 1 antibody [EPR3324] - BSA and Azide free (AB247545)

This data was developed using ab92333, the same antibody clone in a different buffer formulation.

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 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Calpain small subunit 1 antibody [EPR3324] - BSA and Azide free (AB247545)

This data was developed using ab92333, the same antibody clone in a different buffer formulation.

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.

• WB

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Western blot - Anti-Calpain small subunit 1 antibody [EPR3324] - BSA and Azide free (AB247545)

This data was developed using ab92333, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-Calpain small subunit 1 antibody [EPR3324] (<a href='/en-us/products/primary-antibodies/calpain-small-subunit-1-antibody-epr3324-ab92333'>ab92333</a>) at 1/2000 dilution

Lane 1:

T-47D cell lysate at 10 µg

Lane 2:

Fetal brain tissue lysate at 10 µg

Lane 3:

Fetal spleen tissue lysate at 10 µg

Lane 4:

293T (Human embryonic kidney epithelial cell) cell lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 28 kDa

Observed band size: 28 kDa

false

• WB

Lab

Western blot - Anti-Calpain small subunit 1 antibody [EPR3324] - BSA and Azide free (AB247545)

This data was developed using ab92333, the same antibody clone in a different buffer formulation.

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:

Western blot - Anti-Calpain small subunit 1 antibody [EPR3324] (<a href='/en-us/products/primary-antibodies/calpain-small-subunit-1-antibody-epr3324-ab92333'>ab92333</a>)

Predicted band size: 28 kDa

false