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  • 產品名稱：Caspase-6SubstrateVEID-pNA

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Caspase-6SubstrateVEID-pNA
詳情介紹：
Sequence Ac-Val-Glu-Ile-Asp-pNA
Characteristics Ready-to-use colorimetric substrate for caspase-6/Mch2 and related caspases that recognize the amino acid sequence VEID. Caspase-6 and related caspase activity can be quantified by spectrophotometric detection of free pNA (l= 400 nm) after cleavage from the peptide substrate VEID-pNA, using a spectrophotometer or multi-well plate reader.
Purity > 95?% by HPLC
Chemical Name Ac-VEID-pNA, VEID-pNA, Caspase-6 Substrate, Colorimetric
Formula C??H??N?O??
Permeability Not-permeable
Molecular Weight 636.7 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 106 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 VEID- p NA substrates (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: 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).

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).