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  • 产品名称：Caspase-9SubstrateLEHD-pNA

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  • 产品厂商：Biovision
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简单介绍：
Caspase-9SubstrateLEHD-pNA
详情介绍：
Sequence Ac-Leu-Glu-His-Asp-pNA
Characteristics Ready-to-use colorimetric substrate for caspase-9/Mch6 and related caspases that recognize the amino acid sequence LEHD. Caspase-9 and related caspase activity can be quantified by spectrophotometric detection of free pNA (l= 400 nm) after cleavage from the peptide substrate LEHD-pNA, using a spectrophotometer or multi-well plate reader.
Purity > 98 % by HPLC
Chemical Name Ac-LEHD-pNA, Caspase-9 Substrate, colorimetric, LEHD-pNA, LEHDPNA
Formula C₂₉H₃₈N₈O₁₁
Permeability Not-permeable
Molecular Weight 674.7 g/mol
Comment

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 substrate LEHD- p NA (200 µM final conc.) into each tube 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
Product cited in: Lin, Chien, Tseng, Chen, Chang, Lin: "Furano-1,2-Naphthoquinone Inhibits Src and PI3K/Akt Signaling Pathways in Ca9-22 Human Oral Squamous Carcinoma Cells." in: Integrative cancer therapies, Vol. 13, Issue 3, pp. NP18-NP28, 2014 (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).

Cummings, Schnellmann: "Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways." in: The Journal of pharmacology and experimental therapeutics, Vol. 302, Issue 1, pp. 8-17, 2002 (PubMed).

Ghatak, Misra, Toole: "Hyaluronan oligosaccharides inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3-kinase/Akt cell survival pathway." in: The Journal of biological chemistry, Vol. 277, Issue 41, pp. 38013-20, 2002 (PubMed).

Nowak: "Protein kinase C-alpha and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatin-induced apoptosis in renal cells." in: The Journal of biological chemistry, Vol. 277, Issue 45, pp. 43377-88, 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).

Ding, Lin, McGill, Juo, Zhu, Blenis, Yuan, Fisher: "Essential role for caspase-8 in transcription-independent apoptosis triggered by p53." in: The Journal of biological chemistry, Vol. 275, Issue 49, pp. 38905-11, 2001 (PubMed).