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Caspase-3 Colorimetric Assay Kit

Details for Product No. ABIN411828, Supplier: Log in to see
Antigen
  • A830040C14Rik
  • AC-3
  • Apopain
  • Cas3
  • Casp 3
  • Casp3
  • caspase-3
  • Caspase-3
  • caspase 3
  • CC3
  • CG7788
  • CG14902
  • CPP32
  • CPP32B
  • crice
  • Decay
  • DECAY
  • Dmel\\CG7788
  • Dmel\\CG14902
  • drice
  • drIce
  • DrIce
  • DrICE
  • drICE
  • Drice
  • DRICE
  • Drosophila executioner caspase related to Apopain/Yama
  • ICE
  • ice
  • Lice
  • mldy
  • SCA-1
  • xcpp32
  • Yama
Reactivity
Mammalian
19
18
16
7
7
7
5
5
4
4
4
2
2
Application
Biochemical Assay (BCA)
Supplier
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Purpose The Caspase-3/CPP32 Colorimetric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD. The assay is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate DEVD-pNA. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at 400- or 405 nm. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in CPP32 activity.
Sample Type Cell Lysate, Tissue Lysate
Detection Method Colorimetric
Specificity The Caspase-3/CPP32 Colorimetric Assay Kit provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD. The assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA. The pNA light emission can be quantified using a spectrophotometer or a microtiter plate reader at 400- or 405-nm. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in CPP32 activity.
Characteristics Caspase-3,CPP32 Activity Colorimetric Assay Kit: Simple & Convenient Colorimetric Assay to Measure Caspase-3,CPP32 Activity within 1-2 hrs.
Components
  • Cell Lysis buffer
  • 2X Reaction Buffer
  • DEVD-pNA (4 mM)
  • DTT (1 M)
  • Dilution Buffer
Alternative Name Caspase-3 (CASP3 ELISA Kit Abstract)
Background Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells.
Research Area Apoptosis/Necrosis, Autophagy
Application Notes Detection method: Absorbance (400 or 405 nm)
Applications: Detect early/middle stages of apoptosis, differentiate apoptosis from necrosis.
Comment

Absorbance (400 or 405 nm)
Simple one-step procedure, takes 1-2 hours
Fast and convenient
Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in CPP32 activity.

Assay Time 1 - 2 h
Protocol A. General Considerations Aliquot enough 2X Reaction Buffer for the number of assays to be performed. Add DTT to the 2X Reaction Buffer immediately before use (10 mM final concentration: add 10 µL of 1.0 M DTT stock per 1 mL of 2X Reaction Buffer). Protect DEVD-pNA from light. B.
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 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 for immediate assay or aliquot and store at -80 °C for future use.
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 (containing 10 mM DTT) to each sample.
9. Add 5 µL of the 4 mM DEVD-pNA substrate (200 µM final conc.) and incubate at 37 °C for 1-2 hour.
10. 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 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 CPP32 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 CPP32 activity.
Restrictions For Research Use only
Storage -20 °C
Storage Comment Store kit at -20 °C (Store Lysis Buffer, Reaction Buffer, and Dilution Buffer at 4 °C after opening).
Expiry Date 12 months
Supplier Images
Activity Assay (AcA) image for Caspase-3 Colorimetric Assay Kit (ABIN411828) Induction of Caspase-3 Activity by Anti-Fas Antibody in Jurkat-T Cells using Caspase-...
Product cited in: Chandravanshi, Bhonde: "Small molecules exert anti-apoptotic effect and reduce oxidative stress augmenting insulin secretion in stem cells engineered islets against hypoxia." in: European journal of pharmacology, Vol. 791, pp. 424-432, 2016

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Rana, Bera, Bhattacharya, Das, Pan, Das: "Characterization of arsenic-induced cytotoxicity in liver with stress in erythrocytes and its reversibility with Pleurotus florida lectin." in: Toxicology and industrial health, Vol. 31, Issue 2, pp. 108-22, 2015

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