Product name : Caspase_1 Substrate YVAD_pNA
Catalog number : 1104-1000
Quantity: 1000 assays
Supplier name : Biovis
Data sheet : Ask more or other datasheet now !
STORAGE: Store at –20°C, protected from light.
SHELF LIFE: 6 months under proper storage conditions
MOL. WEIGHT: 629.0
PURITY: >98% by HPLC analysis.
Ready-to-use colorimetric substrate for caspase-1/ICE and related caspases that recognize the amino acid sequence YVAD. Caspase-1 and related caspase activity can be quantified by spectrophotometric detection of free pNA (= 400 nm) after cleavage from the peptide substrate YVAD-pNA, using a spectrophotometer or multi-well plate reader. The ready-touse caspase substrate provides an economic alternative for large volume users.
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
Note: Active recombinant human caspase-1 is available to use as a positive control (BioVision, Cat.# 1081-25, -100).
2. Count cells and pellet 1-5 x 106 cells.
3. Resuspend cells in 50 μl of chilled Cell Lysis Buffer (Cat.# 1067-100) 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 100-300 μg protein to 50 μl Cell Lysis Buffer for each assay.
8. Add 50 μl of 2X Reaction Buffer (Cat.# 1068-20, -80) containing 10 mM DTT (Cat.#1201-1) to each sample.
10. Add 5 μl of the 4 mM of YVAD-pNA (200 M final conc.) and incubate at 37°C for 1-2 hour.
9. Read samples at 400- or 405-nm in a microtiter plate reader, or spectrophotometer using a 100-μl micro quartz cuvette (Sigma), or dilute sample to 1 ml with Dilution Buffer (Cat.# 1066-100, -500) and using regular cuvette (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.
For Research Use Only! Not to be used in humans.
Apoptosis Detection Kits & Reagents
Cell Fractionation System
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Monoclonal and Polyclonal Antibodies
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