Product name : Caspase_1 Substrate YVAD_AFC
Catalog number : 1103-1000
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: 719.7
SEQUENCE: Ac-Tyr-Val-Ala-Asp-AFC (AFC, 7-amino-4-trifluoromethyl coumarin)
PURITY: >98% by HPLC analysis.
Ready-to-use fluorometric 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 fluorescent detection of free AFC after cleaved from the peptide substrate YVAD-AFC at Ex. = 400 nm and Em. = 505 nm, using a fluorometer or multi-well 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 volume caspase assays. Cell Lysis Buffer (Cat.#1067-100, -400) and 2X Reaction Buffer (Cat. #1068-20, -80), and DTT used for caspaseassays are also available separately.
1. Induce apoptosis or treat cells by desired method. Concurrently incubate a control culture without treatment.
Note: Active recombinant human caspase-1 is available to use as a positive control (BioVision, Cat.# 1081-25, -100).
2. Pellet 2-5 x 106 cells or use 100-300 μg cell lysates if protein concentration has been measured.
4. Resuspend cells in 50 μl of chilled Cell Lysis Buffer (Cat.# 1067-100).
5. Incubate cells on ice for 10 minutes.
6. Add 50 μl of 2X Reaction Buffer (Cat.# 1068-20,-80) containing 10 mM DTT (Cat.# 1201-1) to each sample.
7. Add 5 μl of the 1 mM YVAD-AFC substrate (50 μM final concentration) and incubate at 37°C for 1-2 hour.
8. Read samples in a fluorometer equipped with a 400-nm excitation and 505-nm emission filters. For a plate-reading set-up, transfer the samples to a 96-well plate. You may also perform the entire assay directly in a 96-well plate. Fold-increase in YVAD-dependent caspase activity can be determined by comparing these results with the level of the untreated control.
For Research Use Only! Not to be used in humans.
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