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Caspase-9 Substrate LEHD-AFC
Functional Studies (Func), Enzyme Activity Assay (EAA)
|19 references available|
|Price||280.50 $ Plus shipping costs $45.00|
|Availability||Will be delivered in 2 to 3 Business Days|
|Description||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 (Cat. #1067-100, - 400) and 2X Reaction Buffer (Cat. #1068-20, -80) and DTT (Cat.# 1201-1) for caspase assays are also available separately.|
|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 (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 LEHD-AFC (50 μM final conc.) into each tube individually and incubate at 37 o 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.|
|Purity||>98% by HPLC analysis.|
|Restrictions||For Research Use only|
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