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Calpain 2 Recombinant Rabbit Monoclonal Antibody [JA43-41]

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Specification

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Catalog# ET1704-90

Calpain 2 Recombinant Rabbit Monoclonal Antibody [JA43-41]

Application

  • WB

  • IF-Cell

  • IHC-P

  • FC

Reactivity

  • Human

Predicted reactivity

  • Mouse

  • Rat

Predicted species support after-sales service
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

Calpain 2 Recombinant Rabbit Monoclonal Antibody [JA43-41]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic peptide within Human Calpain 2 aa 651-700 / 700.

Species Reactivity

Human (Predicted: Mouse, Rat)

Predicted species support after-sales service

Validated Applications

WB, IF-Cell, IHC-P, FC

Molecular Weight

Predicted band size: 80 kDa

Positive Control

HCT 116 cell lysate, MDA-MB-231 cell lysate, 293T cell lysate, HepG2, human lung tissue, human kidney tissue, Hela.

Conjugation

unconjugated

Clone Number

JA43-41

RRID

AB_3070532

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:500-1:2,000

  • IF-Cell

  • 1:50

  • IHC-P

  • 1:50-1:200

  • FC

  • 1:50

Target

Function

Calpain-2 is a heterodimer of a catalytic subunit encoded by CAPN2 gene and a regulatory subunit CAPNS1. The catalytic subunit consists of four domains: protease core 1 domain (PC1), protease core 2 domain (PC2), calpain-type beta-sandwich-like domain (CBSW), and penta EF-hand domain (PEF(L)). The catalytic cleft is formed by PC1 and PC2 upon calcium binding. The catalytic triad consists of residues C105, H262, and N286. Noteworthy, CAPN2 also contains an N-terminal anchor helix, which however is cleaved off upon protease activation. There is no known consensus sequence for calpain-2 proteolysis, but there is evidence for over 130 potential substrates. Proteolytic cleavage by calpain-2 is regulated by presence of Ca2+ ions. It requires supraphysiological (low millimolar) concentration of Ca2+ for activation. Intracellular concentration of Ca2+ (approx. 100 nM) is insufficient for activating calpain-2, so activation occurs upon influx of ions from extracellular space or from endoplasmic reticulum.

Background References

1. Yang X et al. Inhibition of Histone Deacetylase 3 (HDAC3) Mediates Ischemic Preconditioning and Protects Cortical Neurons against Ischemia in Rats. Front Mol Neurosci 9:131 (2016).

2. Jacquemet G et al. L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling. Nat Commun 7:13297 (2016).

Sequence Similarity

Belongs to the peptidase C2 family.

Tissue Specificity

Ubiquitous.

Subcellular Location

Cytoplasm. Cell membrane.

UNIPROT

SwissProt: P17655 Human

SwissProt: O08529 Mouse

SwissProt: Q07009 Rat

Synonyms

Calcium activated neutral proteinase 2 antibody

Calcium activated neutral proteinase antibody

Calcium-activated neutral protease 2, catalytic subunit antibody

Calcium-activated neutral proteinase 2 antibody

CALP80 antibody

Calpain 2 (m/II) large subunit antibody

Calpain 2 catalytic subunit antibody

Calpain 2 large catalytic subunit antibody

Calpain 2 large subunit antibody

Calpain 2, large [catalytic] subunit antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Knockdown (KD)</span><br /><br />Western blot analysis of Calpain 2 on different lysates with Rabbit anti-Calpain 2 antibody (<a href="/products/ET1704-90" style="font-weight: bold;text-decoration: underline;">ET1704-90</a>) at 1/1,000 dilution.<br /><br />Lane 1: HCT 116-si NT cell lysate<br />Lane 2: HCT 116-si Calpain 2 cell lysate<br /><br />Lysates/proteins at 10 µg/Lane.<br /><br />Predicted band size: 80 kDa<br />Observed band size: 75 kDa<br /><br />Exposure time: 40 seconds; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1704-90" style="font-weight: bold;text-decoration: underline;">ET1704-90</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Knockdown (KD)

    Western blot analysis of Calpain 2 on different lysates with Rabbit anti-Calpain 2 antibody (ET1704-90) at 1/1,000 dilution.

    Lane 1: HCT 116-si NT cell lysate
    Lane 2: HCT 116-si Calpain 2 cell lysate

    Lysates/proteins at 10 µg/Lane.

    Predicted band size: 80 kDa
    Observed band size: 75 kDa

    Exposure time: 40 seconds; ECL: K1801;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1704-90) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Western blot analysis of Calpain 2 on different lysates with Rabbit anti-Calpain 2 antibody (<a href="/products/ET1704-90" style="font-weight: bold;text-decoration: underline;">ET1704-90</a>) at 1/1,000 dilution.<br /><br />Lane 1: MDA-MB-231 cell lysate<br />Lane 2: 293T cell lysate<br /><br />Lysates/proteins at 10 µg/Lane.<br /><br />Predicted band size: 80 kDa<br />Observed band size: 70 kDa<br /><br />Exposure time: 1 minutes; ECL: K1802;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1704-90" style="font-weight: bold;text-decoration: underline;">ET1704-90</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of Calpain 2 on different lysates with Rabbit anti-Calpain 2 antibody (ET1704-90) at 1/1,000 dilution.

    Lane 1: MDA-MB-231 cell lysate
    Lane 2: 293T cell lysate

    Lysates/proteins at 10 µg/Lane.

    Predicted band size: 80 kDa
    Observed band size: 70 kDa

    Exposure time: 1 minutes; ECL: K1802;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1704-90) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • ICC staining of Calpain 2 in HepG2 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/ET1704-90" style="font-weight: bold;text-decoration: underline;">ET1704-90</a>, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of Calpain 2 in HepG2 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1704-90, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

  • Immunohistochemical analysis of paraffin-embedded human lung tissue using anti-Calpain 2 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1704-90" style="font-weight: bold;text-decoration: underline;">ET1704-90</a>, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human lung tissue using anti-Calpain 2 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1704-90, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Calpain 2 antibody (<a href="/products/ET1704-90" style="font-weight: bold;text-decoration: underline;">ET1704-90</a>) at 1/200 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1704-90" style="font-weight: bold;text-decoration: underline;">ET1704-90</a>) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Calpain 2 antibody (ET1704-90) at 1/200 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1704-90) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Flow cytometric analysis of Calpain 2 was done on Hela cells. The cells were fixed, permeabilized and stained with the primary antibody (<a href="/products/ET1704-90" style="font-weight: bold;text-decoration: underline;">ET1704-90</a>, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of Calpain 2 was done on Hela cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1704-90, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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