Calpain Activity Fluorometric Assay Kit

Details for Product No. ABIN411643
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Target Name (Antigen)
Synonyms CG18152, CG7563, Dmel\\CG7563, EST C, calpain, Tb08.28A12.350
Reactivity
Mammalian
(1), (3), (2), (1), (1), (1)
Methode Type Sandwich ELISA
Application
Activity Assay (AcA)
Pubmed 19 references available
Quantity 100 tests
Options
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Purpose 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. The Calpain Activity Assay Kit 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.
Sample Type Cell Lysate, Tissue Lysate
Detection Method Fluorometric
Specificity The Calpain Activity Assay Kit 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 (lambda max = 400 nm), upon cleavage of the substrate by calpain, free AFC emits a yellow-green fluorescence (lambda max = 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.
Characteristics Calpain Activity Assay Kit: Rapid, Simple & Convenient Assay to Measure Calpain Activity. Includes Calpain Inhibitor, Z-LLY-FMK. Detection Method: Fluorescence.
Components Extraction Buffer
10X Reaction Buffer
Calpain Substrate Ac-LLY-AFC
Active Calpain I (Positive Control)
Calpain Inhibitor Z-LLY-FMK
Background Activation of calpain is involved in many forms of physiological and pathological processes (e.g., apoptosis). Calpain activation requires cell membrane and Ca 2+, and activated calpain is released into cytosol.
Research Area Enzymes, Metabolism
Application Notes The kit provides a simple and convenient means for analyzing calpain activity in apoptotic and other samples. The assay is based on fluorometric detection of the cleavage of calpain substrate at 505 nm using a fluorometer or a fluorecence plate reader.
Comment

Fluorescence (Ex/Em 400/505 nm)
Simple one-step procedure, takes only ~2 hours
Fast and convenient
The kit contains both positive and negative controls. Active calpain and calpain inhibitor and antibodies are also available separately.

Assay Time 2 h
Protocol 1. Treat cells by desired methods. Concurrently incubate a control culture without treatment.
2. Count cells and pellet approx. 1-2 x 10^6 cells by centrifugation.
3. Resuspend cells in 100 µL Extraction Buffer and incubate samples on ice for 20 minutes. Gently mix the samples by tapping several times during incubation.
4. Centrifuge for 1 min in a microcentrifuge (10,000 g) and transfer supernatant to a fresh tube and put on ice. Assay protein concentration (Note: because of the high reducing agent content in the extraction buffer-dilute about 10-fold then use a Coomassie-based protein assay).
5. Dilute the cell lysate (approx. 50-200 µg) to 85 µL of Extraction Buffer. For positive control, add 1-2 µL Active Calpain to 85 µL of Extraction Buffer. For negative control, use untreated cell lysate or add 1 µL Calpain Inhibitor to the treated cell lysate.
6. Add 10 µL of 10X Reaction Buffer and 5 µL of Calpain Substrate to each assay.
7. Incubate at 37 °C for 1 hour in the dark.
8. Read samples in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. For a plate reading set-up, transfer the samples to a 96-well plate.
9. The changes in calpain activity can be determined by comparing results of treated samples and negative control. Alternatively, the activity can be expressed as Relative Fluorescent Unit (RFU) per milligram protein of each sample.
Restrictions For Research Use only
Storage -80 °C
Storage Comment Store kit at -70 °C (Store Extraction Buffer and 10X Reaction Buffer at 4 °C after opening).
Expiry Date 6-12 months
Product cited in: Wu, Chen, Jiang et al.: "Calpain-dependent cleavage of junctophilin-2 and T-tubule remodeling in a mouse model of reversible heart failure." in: Journal of the American Heart Association, Vol. 3, Issue 3, pp. e000527, 2014 (PubMed).

Chen, Yao, Hasegawa et al.: "Effects of isoproterenol on aquaporin 5 levels in the parotid gland of mice in vivo." in: American journal of physiology. Endocrinology and metabolism, Vol. 306, Issue 1, pp. E100-8, 2014 (PubMed).

McCarthy, Clark, Bartling et al.: "Redox control of the senescence regulator interleukin-1α and the secretory phenotype." in: The Journal of biological chemistry, Vol. 288, Issue 45, pp. 32149-59, 2013 (PubMed).

Lu, Lu, Lai et al.: "Calcium flux and calpain-mediated activation of the apoptosis-inducing factor contribute to enterovirus 71-induced apoptosis." in: The Journal of general virology, Vol. 94, Issue Pt 7, pp. 1477-85, 2013 (PubMed).

Müller, Freed, Dhalla: "Activation of proteases and changes in Na+-K+-ATPase subunits in hearts subjected to ischemia-reperfusion." in: Journal of applied physiology (Bethesda, Md. : 1985), Vol. 114, Issue 3, pp. 351-60, 2013 (PubMed).

Shinde, Sizova, Lin et al.: "ER Stress in Retinal Degeneration in S334ter Rho Rats." in: PLoS ONE, Vol. 7, Issue 3, pp. e33266, 2012 (PubMed).

Liu, Xu, Chase et al.: "Tiam1-regulated osteopontin in senescent fibroblasts contributes to the migration and invasion of associated epithelial cells." in: Journal of cell science, Vol. 125, Issue Pt 2, pp. 376-86, 2012 (PubMed).

Jeong, Shin, Kim et al.: "Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death." in: Journal of experimental & clinical cancer research : CR, Vol. 30, pp. 44, 2011 (PubMed).

Yeh, Kuo, Chan et al.: "Transforming growth factor-β and oxidative stress mediate tachycardia-induced cellular remodelling in cultured atrial-derived myocytes." in: Cardiovascular research, Vol. 91, Issue 1, pp. 62-70, 2011 (PubMed).

Li, Okumura, Nomura et al.: "Oxidatively damaged proteins in the early stage of testicular toxicities in male rats by orally administered with a synthetic oestrogen, diethylstilbestrol." in: Reproductive toxicology (Elmsford, N.Y.), Vol. 31, Issue 1, pp. 26-34, 2011 (PubMed).

Leloup, Shao, Bae et al.: "m-Calpain activation is regulated by its membrane localization and by its binding to phosphatidylinositol 4,5-bisphosphate." in: The Journal of biological chemistry, Vol. 285, Issue 43, pp. 33549-66, 2010 (PubMed).

Huang, Zhou, Saberwal et al.: "Interferon consensus sequence binding protein (ICSBP) decreases beta-catenin activity in myeloid cells by repressing GAS2 transcription." in: Molecular and cellular biology, Vol. 30, Issue 19, pp. 4575-94, 2010 (PubMed).

Tsai, Su, Shyue et al.: "EGb761 ameliorates the formation of foam cells by regulating the expression of SR-A and ABCA1: role of haem oxygenase-1." in: Cardiovascular research, Vol. 88, Issue 3, pp. 415-23, 2010 (PubMed).

Guha, Dey, Dhyani et al.: "Calpain and caspase orchestrated death signal to accomplish apoptosis induced by resveratrol and its novel analog hydroxystilbene-1 [correction of hydroxstilbene-1] in cancer cells." in: The Journal of pharmacology and experimental therapeutics, Vol. 334, Issue 2, pp. 381-94, 2010 (PubMed).

Watterson, Hamilton, Martin et al.: "Urban particulate matter causes ER stress and the unfolded protein response in human lung cells." in: Toxicological sciences : an official journal of the Society of Toxicology, Vol. 112, Issue 1, pp. 111-22, 2009 (PubMed).

Undyala, Dembo, Cembrola et al.: "The calpain small subunit regulates cell-substrate mechanical interactions during fibroblast migration." in: Journal of cell science, Vol. 121, Issue Pt 21, pp. 3581-8, 2008 (PubMed).

Liou, Zhou, Pei et al.: "BimEL up-regulation potentiates AIF translocation and cell death in response to MPTP." in: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 19, Issue 10, pp. 1350-2, 2005 (PubMed).

Song, Li, Du et al.: "Muscle-specific expression of IGF-1 blocks angiotensin II-induced skeletal muscle wasting." in: The Journal of clinical investigation, Vol. 115, Issue 2, pp. 451-8, 2005 (PubMed).

Glading, Bodnar, Reynolds et al.: "Epidermal growth factor activates m-calpain (calpain II), at least in part, by extracellular signal-regulated kinase-mediated phosphorylation." in: Molecular and cellular biology, Vol. 24, Issue 6, pp. 2499-512, 2004 (PubMed).

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Catalog No. ABIN411643
484.00 $
Plus shipping costs $45.00 and $22.00 dry ice
Quantity
Price
100 tests
484.00 $

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