Product name : Caspase-8 Substrate IETD-pNA; Appearance Liquid
Catalog number : 1063-1000
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: 638.6
SEQUENCE: Ac-Ile-Glu-Thr-Asp-pNA (pNA, p-nitroanilide)
PURITY: >95% by HPLC analysis.
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 (= 400 nm) after cleavage from the peptide substrate IETD-pNA, using a spectrophotometer or multi-well plate reader.
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 (Cat.# 1067-100, -400) 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 (Cat.# 1068-20, -80) containing 10 mM DTT (Cat.# 1201-1) to each sample.
9. Add 5 μl of the 4 mM pNA 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 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. 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.
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