Product name : Caspase_2 Substrate VDVAD_AFC
Catalog number : 1071-200
Quantity: 200 assays
Supplier name : Biovis
Data sheet : Ask more or other datasheet now !
STORAGE: Store at –20°C, protected from light. Stable for 6 months
MOL. WEIGHT: 770.7
SEQUENCE: Ac-Val-Asp-Val-Ala-Asp-AFC (AFC, 7-amino-4-trifluoromethyl coumarin)
PURITY: >98% by HPLC analysis
Ready-to-use fluorometric substrate for caspases that recognize the amino acid sequence VDVAD. Caspase activity can be quantified by fluorescent detection of free AFC after cleaved from the peptide substrate VDVAD-AFC at Ex/Em = 400/505 nm using a fluorometer or a fluorescence plate reader. Alternatively, a shift in fluorescence from blue to green upon cleavage can be visualized using a hand-held long-UV lamp. The ready-to-use caspase substrate provides an economic alternative for researchers who perform large amount of caspase assays. Cell Lysis Buffer (Cat.# 1067-100), 2X Reaction Buffer (Cat.# 1068-20), DTT (Cat.# 1201-1), and other reagents used for caspase activity assays are also available separately.
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 or use 50-200 μg cell lysates if protein concentration has been measured.
3. Resuspend cells in 50 μl of chilled Cell Lysis Buffer (Cat.# 1067-100).
4. Incubate cells on ice for 10 minutes.
5. Add 50 μl of 2X Reaction Buffer (Cat.# 1068-20, -80) containing 10 mM DTT (Cat.# 1201-1) to each sample.
6. Add 5 μl of the 1 mM VDVAD-AFC (50 M final conc.) into each tube individually and incubate at 37°C for 1-2 hour.
7. Read samples in a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. For a plate-reading set-up, transfer the samples to a 96-well plate. You may perform the entire assay directly in a 96-well plate. Fold-increase in VDVAD-dependent caspase activity can be determined by comparing these results with the level of the uninduced control.
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