製品の概要

  • 製品名
    Calpain Activity Assay Kit
    Calpain 1 キット 製品一覧
  • サンプルの種類
    Tissue Extracts, Cell Lysate
  • アッセイタイプ
    Quantitative
  • 全工程の試験時間
    2h 00m
  • 種交差性
    交差種: Human
    交差が予測される動物種: Mammal
  • 製品の概要

    Calpain Activity Assay Kit (ab65308) provides optimized buffers and reagents for a convenient measurement of calpain activity. The Extraction Buffer provided with the kit specifically extracts cytosolic proteins without contaminations of cell membrane and lysosome proteases. The Extraction Buffer also prevents auto-activation of calpain during the extraction procedure. Thus, the kit detects only activated calpain in cytosol upon treatment of cells with inducers (e.g., chemicals or drugs). The fluorometric assay is based on the detection of cleavage of calpain substrate Ac-LLY-AFC. Ac-LLY-AFC emits blue light (λmax = 400 nm); upon cleavage of the substrate by calpain, free AFC emits a yellow-green fluorescence (λmax = 505 nm), which can be quantified using a fluorometer or a fluorecence plate reader. Comparison of the fluorescence intensity from a treated sample with a normal control allows determination of the changes in calpain activity.


    Visit our FAQs page for tips and troubleshooting.

  • 特記事項

    Activation of calpain is involved in many forms of physiological and pathological processes (e.g., apoptosis). Calpain activation requires cell membrane and Ca²+, and activated calpain is released into cytosol.

     

    If additional Ac-LLY-AFC substrate is needed, it can be purchased separately as ab171379.

  • アプリケーション
    適用あり: Functional Studiesmore details

法規制情報

製品の特性

  • 保存方法
    Store at -80°C. Please refer to protocols.
  • 内容 ラベル 100 tests
    10X Reaction Buffer Clear 1 x 1.5ml
    Active Calpain I (Positive Control) Green 1 x 10µl
    Calpain Inhibitor Z-LLY-FMK Orange 1 x 10µl
    Calpain Substrate Ac-LLY-AFC Amber 1 x 500µl
    Extraction Buffer WM 1 x 25ml
  • 研究分野
  • 機能
    Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction.
  • 組織特異性
    Ubiquitous.
  • 配列類似性
    Belongs to the peptidase C2 family.
    Contains 1 calpain catalytic domain.
    Contains 4 EF-hand domains.
  • 細胞内局在
    Cytoplasm. Cell membrane. Translocates to the plasma membrane upon Ca(2+) binding.
  • Information by UniProt
  • 別名
    • Ca2 activated neutral protease
    • Calcium activated neutral proteinase
    • Calcium activated neutral proteinase small subunit
    • Calcium dependent protease small subunit
    • Calcium dependent protease small subunit 1
    • Calcium-activated neutral proteinase 1
    • Calpain 1
    • Calpain 1 large subunit
    • Calpain 1, (mu/I) large subunit
    • Calpain mu type
    • Calpain mu-type
    • Calpain regulatory subunit
    • Calpain small subunit 1
    • Calpain, large polypeptide L1
    • Calpain-1 catalytic subunit
    • Calpain-1 large subunit
    • CAN1_HUMAN
    • CANP
    • CANP 1
    • CANP small subunit
    • CANP1
    • CANPL 1
    • CANPL1
    • CAPN 1
    • CAPN1
    • Cell proliferation inducing protein 30
    • Cell proliferation-inducing gene 30 protein
    • Micromolar Calpain
    • Micromolar-calpain
    • Mu Calpain
    • muCANP
    • muCL
    see all
  • 参照データベース

関連製品

アプリケーション

Our Abpromise guarantee covers the use of ab65308 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
Functional Studies Use at an assay dependent concentration.

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  • Different amounts of positive control (Calpain I) treated with 1 μL of inhibitor (Z-LLY-FMK), background signal subtracted, duplicates; +/- SD.

  • 10e7 Jurkat cells (in 10 mL) were cultured in the absence or presence of 10 μM Camptothecin (CPT) (ab120115) or 10 μg/mL Cycloheximide (CHX) (ab120093) for 4 hours. Pelleted cells were lysed in 0.5 mL of Extraction Buffer and tested directly. Background signal subtracted, duplicates; +/- SD.

  • Typical Data for ab65308: Active Calpain (1 µg) was incubated at 37 °C for one hour using the Calpain Substrate with or without 20 µM Calpain Inhibitor.

プロトコール

参考文献

This product has been referenced in:
  • Benusa SD  et al. Acute neuroinflammation induces AIS structural plasticity in a NOX2-dependent manner. J Neuroinflammation 14:116 (2017). Read more (PubMed: 28595650) »
  • Wert KJ  et al. CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model. Hum Mol Genet 24:4584-98 (2015). Functional Studies . Read more (PubMed: 25994508) »

See all 11 Publications for this product

レビューと Q&A

There is ˜50 mM Calcium in the 10X reaction buffer but not in Extraction buffer. The Extraction buffer however is supplied with optimal concentrations of EDTA and EGTA to chelate the calcium to avoid any autoactivation of calpain during the extraction ...

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Ab65308 measures overall calpain activity. It will measure activity from any active Calpain isoform in the sample. Calpain I and II are mainly expressed in the central nervous system. So depending on what your sample is, the calpain isoform can differ....

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For the Calpain assay we highly recommend using fresh tissues or tissue samples snap-frozen after collection. The frozen tissue can be ground and then homogenized in the extraction buffer.

One point to note is : Inactive calpain remains a...

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The success of this kit with frozen samples depends on how well the samples were frozen, for how long they had been frozen, if they underwent repeated freeze thaw and so on.

Immediate freezing of the sample in aliquots and storing ...

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This kit has the substrate bound to the AFC fluorophore.

The success of this kit with frozen samples depends on how well the samples were frozen, for how long they had been frozen, if they underwent repeated freeze thaw and so on.
Immediate freezing of the sample in aliquots and storing for up to 2 mont...

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I can confirm that for the sample preparation the cells will not need to be trypsinized. They can be scraped from the flask after after adding the extraction buffer and incubated, but keep in mind that this would dilute the sample for the activity assa...

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This Calpain Activity Assay Kit includes Active Calpain I as positive control.

The Calpain protein provided in ab65308 is a native protein. We pack 14.5 ug of Calpain/tube in this kit.

Thank you once again for your enquiries.

I have obtained some further information for you.

Unfortunately, I cannot disclose the contents of the extraction buffer due to proprietary restrictions, but I can assure you that it is str...

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1-10 of 26 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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