Caspase-12 Fluorometric Assay Kit

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Antigen
  • Caspase-12
  • caspase 12
  • Casp12
  • CASP12
Reactivity
Mammalian
9
7
6
2
2
2
2
1
1
1
1
1
1
Application
Biochemical Assay (BCA)
Options
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Purpose The Caspase-12 Fluorometric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence ATAD. The assay is based on detection of cleavage of substrate ATAD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin). ATAD-AFC emits blue light (γmax = 400 nm), upon cleavage of the substrate by caspase-12 or related caspases, free AFC emits a yellow-green fluorescence (λmax = 505 nm), which can be quantified using a fluorometer or a fluorecence microtiter plate reader.
Sample Type Cell Lysate, Tissue Samples
Detection Method Fluorometric
Specificity The Caspase-12 Fluorometric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence ATAD. The assay is based on detection of cleavage of substrate ATAD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin). ATAD-AFC emits blue light (lambda max = 400 nm), upon cleavage of the substrate by caspase-12 or related caspases, free AFC emits a yellow-green fluorescence (lambda max = 505 nm), which can be quantified using a fluorometer or a fluorecence microtiter plate reader. Comparison of the fluorescence of AFC from an apoptotic sample with an uninduced control allows determination of the fold increase in caspase-12 activity.
Characteristics Caspase-12 Activity Fluorometric Assay Kit: Simple & Convenient Fluorometric Assay to Measure Caspase-12 Activity within 1-2 hrs.
Components Cell Lysis Buffer
2X Reaction Buffer
ATAD-AFC Substrate
DTT (1 M)
Target Name (Antigen) Caspase 12 (CASP12)
Alternative Name Caspase-12 (CASP12 ELISA Kit Abstract)
Background Caspase family of proteases are the central mediators of apoptosis in mammalian cells. The Caspase-12 Fluorometric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence ATAD. The assay is based on detection of cleavage of substrate ATAD-AFC (AFC: 7-amino-4-trifluoromethyl coumarin). ATAD-AFC emits blue light (γmax = 400 nm), upon cleavage of the substrate by caspase-12 or related caspases, free AFC emits a yellow-green fluorescence (λmax = 505 nm), which can be quantified using a fluorometer or a fluorecence microtiter plate reader.
Application Notes Detect early/middle stages of apoptosis, differentiate apoptosis from necrosis.
Comment

Further details regarding sample type: Cell and tissue lysates

Assay Time 1 - 2 h
Protocol A. General Considerations Aliquot enough 2X Reaction Buffer for the number of assays to be performed. Add DTT to the 2X Reaction Buffer immediately before use (10 mM final concentration: add 10 µL of 1.0 M DTT stock per 1 mL of 2X Reaction Buffer). After thawing, store the Cell Lysis Buffer and 2X Reaction Buffer at 4 °C. Protect ATAD-AFC from light. B.
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 2-5 x 10^6 cells or use 100-300 µg cell lysates if protein concentration has been measured.
3. Resuspend cells in 50 µL of chilled Cell Lysis Buffer. Incubate on ice for 10 min.
4. Add 50 µL of 2X Reaction Buffer (containing 10 mM DTT) to each sample.
6. Add 5 µL of the ATAD-AFC substrate (50 µM final concentration) and incubate at 37 °C for 1-2 hour.
7. 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. You may also perform the entire assay directly in a 96-well plate. Fold-increase in caspase-12 activity can be determined by comparing these results with the level of the uninduced control.
Restrictions For Research Use only
Storage -20 °C
Storage Comment Store kit at -20 °C (Store Cell Lysis Buffer & 2X Reaction Buffer at 4 °C after opening).
Expiry Date 6-12 months
Product cited in: Okle, Stemmer, Deschl, Dietrich: "L-BMAA induced ER stress and enhanced caspase 12 cleavage in human neuroblastoma SH-SY5Y cells at low nonexcitotoxic concentrations." in: Toxicological sciences : an official journal of the Society of Toxicology, Vol. 131, Issue 1, pp. 217-24, 2013 (PubMed).

Trisciuoglio, Uranchimeg, Cardellina, Meragelman, Matsunaga, Fusetani, Del Bufalo, Shoemaker, Melillo: "Induction of apoptosis in human cancer cells by candidaspongiolide, a novel sponge polyketide." in: Journal of the National Cancer Institute, Vol. 100, Issue 17, pp. 1233-46, 2008 (PubMed).

Neekhra, Luthra, Chwa, Seigel, Gramajo, Kuppermann, Kenney: "Caspase-8, -12, and -3 activation by 7-ketocholesterol in retinal neurosensory cells." in: Investigative ophthalmology & visual science, Vol. 48, Issue 3, pp. 1362-7, 2007 (PubMed).

Peyrou, Hanna, Cribb: "Cisplatin, gentamicin, and p-aminophenol induce markers of endoplasmic reticulum stress in the rat kidneys." in: Toxicological sciences : an official journal of the Society of Toxicology, Vol. 99, Issue 1, pp. 346-53, 2007 (PubMed).

Kikuchi, Almer, Yamashita, Guégan, Nagai, Xu, Sosunov, McKhann, Przedborski: "Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, Issue 15, pp. 6025-30, 2006 (PubMed).

Zhou, Pandak, Lyall, Natarajan, Hylemon: "HIV protease inhibitors activate the unfolded protein response in macrophages: implication for atherosclerosis and cardiovascular disease." in: Molecular pharmacology, Vol. 68, Issue 3, pp. 690-700, 2005 (PubMed).

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