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Index / Biovis / Caspase-3 Substrate DEVD-pNA; Appearance Liquid / Product Detail : 1008-200 Caspase-3 Substrate DEVD-pNA; Appearance Liquid
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Sep
29th

#1008-200 Caspase-3 Substrate DEVD-pNA; Appearance Liquid

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  Price : 379   EUR
431   USD
294   GBP
1595   Zloty
50814   JPY
2928   NOK
3138   SEK
429   CHF

Product name : Caspase-3 Substrate DEVD-pNA; Appearance Liquid

Catalog number : 1008-200

Quantity: 200 assays

Availability: Yes

Supplier name : Biovis

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More Details about

STORAGE CONDITIONS: Store at –20°C, protected from light.
SHELF LIFE: 6 months under proper storage conditions
MOLECULAR WEIGHT: 638.58
SEQUENCE: Ac-Asp-Glu-Val-Asp-pNA
PURITY: >98% by HPLC analysis


DESCRIPTION:
Ready-to-use colorimetric substrate for CPP32/caspase-3 and related caspases that recognize the amino acid sequence DEVD. The sequence DEVD is based on caspase-3 cleavage site in poly (ADP-ribose) polymerase (PARP). CPP32 and related caspase activity can be quantified by spectrophotometric detection of free pNA (λ = 400 nm) after cleavage from the peptide substrate DEVD-pNA, using a spectrophotometer or multi-well plate reader. The ready-to-use caspase substrate provides an economic alternative for researchers who perform large amounts of caspase assays.


ASSAY PROCEDURE:
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 50-200 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 DEVD-pNA (200 μM final conc.) and incubate at 37°C for 1-2 hour.
9. Read samples at 400- or 405-nm in a microtiter plate reader, or spectrophotometer using a 100-μl micro quartz cuvet (Sigma), or dilute sample to 1 ml with Dilution Buffer (Cat.#1066-100, -500) and using regular cuvet (note: Dilution of the samples proportionally decreases the reading).

You may also perform the entire assay directly in a 96-well plate.
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.

 

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