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Caspase colorimetric substrate set plus

 
ALX-850-274-KI01 1 Set 843.00 USD
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Product Specification

Applications:Colorimetric detection
 
Kit/Set Contains:Contains the following components:  
- Caspase-1 Substrate, Ac-YVAD-pNA (125µl)  
- Caspase-2 Substrate, Ac-VDVAD-pNA (125µl)  
- Caspase-3 Substrate, Ac-DEVD-pNA (125µl)  
- Caspase-5 Substrate, Ac-WEHD-pNA (125µl)  
- Caspase-6 Substrate, Ac-VEID-pNA (125µl)  
- Caspase-8 Substrate, Ac-IETD-pNA (125µl)  
- Caspase-9 Substrate, Ac-LEHD-pNA (125µl)  
- Cell Lysis Buffer (100ml)  
- Dilution Buffer (160ml)  
- 2x Reaction Buffer (20ml)  
- DTT (1M) (400µl)
 
Quantity:7x25 tests
 
Handling:For optimal storage temperature of single components please refer to individual label. Avoid freeze/thaw cycles.
 
Shipping:Shipped on Blue Ice
 
Short Term Storage:-20°C
 
Protocol:Assay Procedure:
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture without induction.
2. Count cells and pellet 1-5x106 cells.
3. Resuspend cells in 50 µl of chilled Cell Lysis Buffer and incubate cells on ice for 10 min. Centrifuge for 1 min in a microcentrifuge (10’000 x g).
4. Transfer supernatant to a fresh tube and assay protein concentration.
5. Dilute 100-300 µg protein to 50 µl Cell Lysis Buffer for each test.
6. Add 50 µl of 2x Reaction Buffer containing 10 mM DTT to each sample.
7. Add 5 µl of the 4 mM pNA conjugated substrates (200 µM final conc.) into each tube individually and incubate at 37°C for 1-2 hours.
8. Read samples at 400 or 405nm in a microtiter plate reader, or spectrophotometer using a 100 µl micro quartz cuvet (Sigma), or dilute sample to 1ml with Dilution Buffer and using regular cuvet (note: dilution of the samples proportionally decreases the reading).

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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