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  • 產(chǎn)品名稱：Caspase-8SubstrateIETD-pNA

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  • 產(chǎn)品廠商：Biovision
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Caspase-8SubstrateIETD-pNA
詳情介紹：
Sequence Ac-Ile-Glu-Thr-Asp-pNA
Purity > 95?% by HPLC
Chemical Name Ac-IETD-pNA, Caspase-8 Substrate, Colorimetric
Formula C??H??N?O??
Permeability Not-permeable
Molecular Weight 638.6 g/mol
Comment

Ready-to-use colorimetric substrate for FLICE/caspase-8 and related caspases that recognize the amino acid sequence IETD. The sequence IETD is based on caspase-8 cleavage site in CPP32/Caspase-3 proenzyme. FLICE and related caspase activity can be quantified by spectrophotometric detection of free pNA (l= 400 nm) after cleavage from the peptide substrate IETD-pNA, using a spectrophotometer or multi-well plate reader.
Note: Background reading from cell lysates and buffers should be subtracted from the readings of both induced and the uninduced samples before calculating fold increase in caspase activity.
Protocol 1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 1-5 x 10^6 cells.
3. Resuspend cells in 50 μL of chilled Cell Lysis Buffer and incubate cells on ice for 10 minutes.
4. Centrifuge for 1 min in a microcentrifuge (10,000 x g).
5. Transfer supernatant (cytosolic extract) to a fresh tube and put on ice.
6. Assay protein concentration.
7. Dilute 50-200 μg protein to 50 μL Cell Lysis Buffer for each assay.
8. Add 50 μL of 2X Reaction Buffer containing 10 mM DTT to each sample.
9. Add 5 μL of the 4 mM p NA conjugated substrates (200 μM final conc.) into each tube individually and incubate at 37 °C for 1-2 hour.
10. Read samples at 400 or 405-nm in a microtiter plate reader, or spectrophotometer using a 100- μL micro quartz cuvet (Sigma), or dilute sample to 1 mL with Dilution Buffer and using regular cuvet (note: Dilution of the samples proportionally decreases the reading). You may also perform the entire assay directly in a 96-well plate. Fold-increase in caspase activity can be determined by comparing these results with the level of the uninduced control. Note: Background reading from cell lysates and buffers should be subtracted from the readings of both induced and the uninduced samples before calculating fold increase in caspase activity.
Restrictions For Research Use only
Format Liquid
Handling Advice Protect from light and moisture
Storage -20 °C
Expiry Date 12 months
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Yuan, Fu, Wang, Shi, Huang, Song, Li, Song, Luo: "Voltage-dependent anion channel 1 is involved in endostatin-induced endothelial cell apoptosis." in: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 22, Issue 8, pp. 2809-20, 2008 (PubMed).

Lee, Cherla, Tesh: "Simultaneous induction of apoptotic and survival signaling pathways in macrophage-like THP-1 cells by Shiga toxin 1." in: Infection and immunity, Vol. 75, Issue 3, pp. 1291-302, 2007 (PubMed).

Lieman, Worley, Harbour: "Loss of Rb-E2F repression results in caspase-8-mediated apoptosis through inactivation of focal adhesion kinase." in: The Journal of biological chemistry, Vol. 280, Issue 11, pp. 10484-90, 2005 (PubMed).

Ishigami, Fujita, Handa, Shirasawa, Koseki, Kitamura, Enomoto, Sato, Shimosawa, Maruyama: "Senescence marker protein-30 knockout mouse liver is highly susceptible to tumor necrosis factor-alpha- and Fas-mediated apoptosis." in: The American journal of pathology, Vol. 161, Issue 4, pp. 1273-81, 2002 (PubMed).

Shain, Landowski, Dalton et al.: "Adhesion-mediated intracellular redistribution of c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long confers resistance to CD95-induced apoptosis in hematopoietic ..." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 168, Issue 5, pp. 2544-53, 2002 (PubMed).

Kawakami, Kawakami, Puri: "Intratumor administration of interleukin 13 receptor-targeted cytotoxin induces apoptotic cell death in human malignant glioma tumor xenografts." in: Molecular cancer therapeutics, Vol. 1, Issue 12, pp. 999-1007, 2002 (PubMed).

Vu, Bortner, Cidlowski: "Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death." in: The Journal of biological chemistry, Vol. 276, Issue 40, pp. 37602-11, 2001 (PubMed).

Varadhachary, Edidin, Hanlon, Peter, Krammer, Salgame: "Phosphatidylinositol 3'-kinase blocks CD95 aggregation and caspase-8 cleavage at the death-inducing signaling complex by modulating lateral diffusion of CD95." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 166, Issue 11, pp. 6564-9, 2001 (PubMed).

Doostzadeh-Cizeron, Yin, Goodrich: "Apoptosis induced by the nuclear death domain protein p84N5 is associated with caspase-6 and NF-kappa B activation." in: The Journal of biological chemistry, Vol. 275, Issue 33, pp. 25336-41, 2000 (PubMed).

Gómez-Angelats, Bortner, Cidlowski: "Protein kinase C (PKC) inhibits fas receptor-induced apoptosis through modulation of the loss of K+ and cell shrinkage. A role for PKC upstream of caspases." in: The Journal of biological chemistry, Vol. 275, Issue 26, pp. 19609-19, 2000 (PubMed).

Sawa, Wiegand, Cooper, Margolis, Sharp, Lawler, Greenamyre, Snyder, Ross: "Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization." in: Nature medicine, Vol. 5, Issue 10, pp. 1194-8, 1999 (PubMed).