概述

  • 产品名称
    Calpain Activity Assay试剂盒
    参阅全部 Calpain 1 试剂盒
  • 样品类型
    Tissue Extracts, Cell Lysate
  • 检测类型
    Quantitative
  • 检测时间
    2h 00m
  • 种属反应性
    与反应: Mammals, Other species
  • 产品概述

    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.

  • 说明

    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.

性能

  • 存放说明
    Please refer to protocols.
  • 组件 标识符 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
  • 研究领域
  • 功能
    Calcium-regulated non-lysosomal thiol-protease which catalyze limited proteolysis of substrates involved in cytoskeletal remodeling and signal transduction.
  • 组织特异性
    Ubiquitous.
  • 序列相似性
    Belongs to the peptidase C2 family.
    Contains 1 calpain catalytic domain.
    Contains 4 EF-hand domains.
  • 细胞定位
    Cytoplasm. Cell membrane. Translocates to the plasma membrane upon Ca(2+) binding.
  • Information by UniProt
  • 别名
    • 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

相关产品

图片

  • 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.

实验方案

文献

This product has been referenced in:
  • Wolhuter K  et al. Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation. Mol Cell 69:438-450.e5 (2018). Functional Studies . Read more (PubMed: 29358077) »
  • Pahima H  et al. Hypoxic-induced truncation of voltage-dependent anion channel 1 is mediated by both asparagine endopeptidase and calpain 1 activities. Oncotarget 9:12825-12841 (2018). Read more (PubMed: 29560113) »
See all 24 Publications for this product

客户评价及客户问答

1-10 of 27 Abreviews or Q&A

Question
Answer

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. Also, Calpain I and II (also called mu-calpain and m-calpain respectively) are activated by micro- and nearly millimolar concentrations of Ca2+ within the cell. To determine Calpain I activity exclusively either you have to make sure that the calcium concentration is in micromolar range in the sample or you have to purify calpain I specifically to test its activity.

Read More
Abreviews
Calpain activity kit tested in rat's gastrocnemius (fast fibers). 2 conditions: control and 7 days of sepsis.
10mg of muscle was crushed with ultraturax on ice in the extraction buffer of the kit. After a centrifugation (4min, 4°C, 11000rpm) proteins were quantify in supernatant by a Bradford test. The kit was made according to the instructions, samples in duplicate and read at 405/510nm.
the figure explain the % of variation of calpain activity in septic muscle compare to control group.
No difference was observed

Dr. Florine Tissier

Verified customer

提交于 Feb 12 2018

Answer

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 procedure.

We do not provide separate source of calcium other than what is present in buffer.

Read More

Answer


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 associated to the cell membrane and active calpain is released into the cytosol. Using an extraction buffer which pokes holes in the cell membrane only the active calpain in the cytosol will leak out and get assayed. 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). Any cell lysis buffer will not distinguish between inactive and active calpain. When using a tissue homogenate there might be some autoactivation of calpain.

Read More

Answer



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 months without any freeze thaw is ideal.

The lab also confirmed that more than 1-2% DMSO will interfere with the assay.

Read More

Question
Answer

This kit has the substrate bound to the AFC fluorophore.

Read More

Answer

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 months without any freeze thaw is ideal.

Read More

Answer

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 assay. It would require a few mls of the extraction buffer to cover the cells. An alternative is to wash the cells with PBS. Then scrape the cells in PBS, spin it down, remove PBS and resuspend cells in extraction buffer.

Read More

Question
Answer

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

Read More

Answer

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

Read More

1-10 of 27 Abreviews or Q&A

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

注册