Caspase-9 Substrate LEHD-AFC

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Application
Enzyme Activity Assay (EAA), Functional Studies (Func)
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Sequence Ac-Leu-Glu-His-Asp-AFC
Characteristics Ready-to-use fluorometric substrate for caspase-9/Mch-6 and related caspases that recognize the amino acid sequence LEHD. Caspase activity can be quantified by fluorescent detection of free AFC after cleavage from the peptide substrate LEHD-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 amount of caspase assays. Cell Lysis Buffer for caspase assays are also available separately.
Purity > 98 % by HPLC
Chemical Name Ac-LEHD-AFC, Caspase-9 Substrate, Fluorogenic
Formula C₃₃H₃₈F₃N₇O₁₁
Permeability Not-permeable
Molecular Weight 765.7 g/mol
Protocol 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 containing 10 mM DTT to each sample.
6. Add 5 µL of the 1 mM LEHD-AFC (50 µM 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 LEHD-dependent activity can be determined by comparing these results with the level of the uninduced control.
Restrictions For Research Use only
Format Liquid
Handling Advice Protect from light and moisture
Storage -20 °C
Expiry Date 12 months
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Coletti, Yang, Marazzi, Sassoon: "TNFalpha inhibits skeletal myogenesis through a PW1-dependent pathway by recruitment of caspase pathways." in: The EMBO journal, Vol. 21, Issue 4, pp. 631-42, 2002 (PubMed).

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Vu, Bortner, Cidlowski: "Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death." in: The Journal of biological chemistry, Vol. 276, Issue 40, pp. 37602-11, 2001 (PubMed).

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