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AB266628

Human CAPN2 (Calpain 2) knockout HEK-293T cell line

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CAPN2 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 17 bp deletion in exon 4 and 1 bp insertion in exon 4 and 47 bp deletion in exon 4 and 8 bp deletion in exon 4.

View Alternative Names

CALP80, CAN2_HUMAN, CANP 2, CANP L2, CANPml, CAPN 2, Calcium activated neutral proteinase, Calcium-activated neutral protease 2, catalytic subunit, Calcium-activated neutral proteinase 2, Calpain 2 (m/II) large subunit, Calpain 2 large catalytic subunit, Calpain 2, large [catalytic] subunit, Calpain M-type, Calpain large polypeptide L2, Calpain-2 catalytic subunit, Calpain-2 large subunit, Calpain2, Capa2, FLJ39928, M calpin, M type, M-calpain, Millimolar-calpain, mCANP

6 Images
Western blot - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)
  • WB

Lab

Western blot - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)

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° 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] (<a href='/en-us/products/primary-antibodies/calpain-2-antibody-epr2562y-ab75994'>ab75994</a>) 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 (ab266628)

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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Western blot - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)
  • WB

Lab

Western blot - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)

Lanes 1-4 : Merged signal (red and green). Green - ab126600 observed at 80 kDa. Red - loading control ab8245 observed at 36 kDa.

ab126600 Anti-Calpain 2 antibody [EPR5977] was shown to specifically react with Calpain 2 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266628 (knockout cell lysate ab257379) was used. Wild-type and Calpain 2 knockout samples were subjected to SDS-PAGE. ab126600 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Calpain 2 antibody [EPR5977] (<a href='/en-us/products/primary-antibodies/calpain-2-antibody-epr5977-ab126600'>ab126600</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

CAPN2 knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (ab266628)

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

LNCaP cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 80 kDa

Observed band size: 80 kDa

false

Sanger Sequencing - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)
  • Sanger seq

Unknown

Sanger Sequencing - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)

Allele-4 : 1 bp insertion in exon 4.

Sanger Sequencing - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)
  • Sanger seq

Unknown

Sanger Sequencing - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)

Allele-2 : 17 bp deletion in exon 4.

Sanger Sequencing - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)
  • Sanger seq

Unknown

Sanger Sequencing - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)

Allele-3 : 8 bp deletion in exon 4.

Sanger Sequencing - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)
  • Sanger seq

Unknown

Sanger Sequencing - Human CAPN2 (Calpain 2) knockout HEK-293T cell line (AB266628)

Allele-1 : 47 bp deletion in exon 4

Key facts

Cell type

HEK-293T

Species or organism

Human

Tissue

Kidney

Form

Liquid

form

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, 17 bp deletion in exon 4 and 1 bp insertion in exon 4 and 47 bp deletion in exon 4 and 8 bp deletion in exon 4

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Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Reactivity", "Dilution Info", "Notes"] }, "values": { "WB": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }
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Product details

Recommended control: Human wild-type HEK293T cell line (ab255449). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.

We will provide viable cells that proliferate on revival.

This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.

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What's included?

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Properties and storage information

Gene name
CAPN2
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Shipped at conditions
Dry Ice
Appropriate short-term storage conditions
-196°C
Appropriate long-term storage conditions
-196°C
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Handling procedures

Initial handling guidelines

Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.

1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.

Subculture guidelines
  • All seeding densities should be based on cell counts gained by established methods.
  • A guide seeding density of 2x104 cells/cm2 is recommended.
  • Cells should be passaged when they have achieved 80-90% confluence.
Culture medium

DMEM (High Glucose) + 10% FBS

Cryopreservation medium

Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

Calpain 2 also known as CAPN2 or calpain-2 is a heterodimer protease enzyme weighing around 80 kDa. It belongs to the calpain family which requires calcium ions for activation. Calpain 2 expresses in many tissues including the brain muscles and liver indicating its widespread role in cellular processes. The enzyme comprises a large catalytic subunit and a small regulatory subunit each encoded by different genes.
Biological function summary

Calpain-2 plays a significant role in cell signaling cytoskeletal remodeling and apoptosis regulation. It does not typically function in isolation but partners with other molecules forming complexes necessary for precise proteolytic activity. This enzyme modulates several cell functions by cleaving substrates like cytoskeletal proteins transcription factors and enzymes which allows cells to respond adaptively to various stimuli.

Pathways

Calpain-2 integrates into important signaling cascades such as the ERK and PI3K/AKT pathways. It influences these pathways by processing specific proteins and modifying their activity thereby affecting cell survival and proliferation. Calpain-2 closely interacts with proteins like phosphatidylinositol-45-bisphosphate 3-kinase (PI3K) and extracellular signal-regulated kinases (ERKs) within these pathways emphasizing its importance in finely-tuning cellular responses.

Calpain-2 links to several pathological conditions including neurodegenerative diseases like Alzheimer's and muscular disorders such as Duchenne Muscular Dystrophy. In Alzheimer's disease calpain-2 activity influences the processing of amyloid precursor proteins contributing to plaque formation. In Duchenne Muscular Dystrophy calpain-2 activity disrupts muscle fiber integrity through its interaction with proteins like dystrophin aggravating the disorder's progression.
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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 2 US: 2

Adherent/suspension

Adherent

Gender

Female

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Product protocols

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Target data

See full target information CAPN2
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Complementary Products

Primary Antibodies

AB126600

Anti-Calpain 2 antibody [EPR5977]

RabMAb|Recombinant|KO Validated

Western blot - Anti-Calpain 2 antibody [EPR5977] (AB126600)

  • 0
  • 1
Primary Antibodies

AB108400

Anti-Calpain 1 antibody [EPR3319]

RabMAb|Recombinant|KO Validated

Western blot - Anti-Calpain 1 antibody [EPR3319] (AB108400)

  • 4
  • 1
Biochemicals

AB141464

PD 150606, Calpain inhibitor

Chemical Structure - PD 150606, Calpain inhibitor (AB141464)

  • 5
  • 1
Primary Antibodies

AB181115

Anti-Raf1 antibody [EP4969] - N-terminal

KO Validated|RabMAb|Recombinant|Lab Essentials

Immunocytochemistry/ Immunofluorescence - Anti-Raf1 antibody [EP4969] - N-terminal (AB181115)

  • 0
  • 0

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