Product name : Caspase_10 Substrate AEVD_pNA
Catalog number : 1114-200
Quantity: 200 assays
Supplier name : Biovis
Data sheet : Ask more or other datasheet now !
STORAGE CONDITIONS: Store at –20°C, protected from light.
SHELF LIFE: 6 months under proper storage conditions
MOLECULAR WEIGHT: 594.57
PURITY: >99% by HPLC analysis.
Ready-to-use colorimetric substrate for caspases that recognize the amino acid sequence AEVD. Caspase activity can be quantified by spectrophotometric detection of free pNA ( =400 nm) after cleaved from the peptide substrate AEVD-pNA, using a spectrophotometer or multi-well plate reader. The ready-to-use caspase substrate provides an economical alternative for researchers who perform large volume of caspase assays.
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) 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 100-200 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 of AEVD-pNA (200 μM final conc.) 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).
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.
For Research Use Only! Not to be used in humans.
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