Anti-Calpain 2 antibody [EPR2562Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Calpain 2 antibody [EPR2562Y] (AB75994)

Intracellular flow cytometric analysis of permeabilized HeLa cells using ab75994 (red) or a rabbit IgG (negative) (green).

• WB

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Western blot - Anti-Calpain 2 antibody [EPR2562Y] (AB75994)

All lanes:

Western blot - Anti-Calpain 2 antibody [EPR2562Y] (ab75994) at 1/500 dilution

All lanes:

HeLa cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 80 kDa

Observed band size: 75 kDa

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• WB

Collaborator

Western blot - Anti-Calpain 2 antibody [EPR2562Y] (AB75994)

ab75994 was shown to react with CAPN2 in wild-type MDA-MB-231 (wpxld GFP) cells in Western blot with loss of signal observed in a CAPN2 knockout cell line. Wild-type MDA-MB-231 (wpxld GFP) and CAPN2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab75994 overnight at 4 °C at a 1/2000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2 μg/ml before imaging. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Calpain 2 antibody [EPR2562Y] (ab75994)

Predicted band size: 80 kDa

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• WB

Lab

Western blot - Anti-Calpain 2 antibody [EPR2562Y] (AB75994)

Lanes 1 - 4 : Merged signal (red and green). Green - ab75994 observed at 75 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab75994 was shown to react with Calpain 2 in wild-type HEK-293T cells in Western blot with loss of signal observed in CAPN2 knockout cell line ab266628 (CAPN2 knockout cell lysate ab257379). Wild-type HEK-293T and CAPN2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab75994 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Calpain 2 antibody [EPR2562Y] (ab75994) at 1/2000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 40 µg

Lane 2:

CAPN2 knockout HEK-293T cell lysate at 40 µg

Lane 2:

Western blot - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-capn2-calpain-2-knockout-hek-293t-cell-line-ab266628'>ab266628</a>)

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

LNCaP cell lysate at 20 µg

Predicted band size: 80 kDa

Observed band size: 75 kDa

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