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Caspase-8 Colorimetric Assay Kit K113-100 100 assays biovision
اعتبار قیمت 95.3.1
لطفا پس از پایان اعتبار قیمت با تلفن 88920648 تماس حاصل فرمائید
Kit Summary:
• Detection method- Absorbance (400 or 405 nm)
• Sample type- Cell and tissue lysates
• Species reactivity- Mammalian
• Kit size- Convenient sizes (25, 100, 200, 400 assays)
• Applications- Detect early/middle stages of apoptosis; differentiate apoptosis from necrosis.
Features & Benefits:
• 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 FLICE
activity.
Kit components:
• Cell Lysis Buffer
• 2X Reaction Buffer
• IETD-pNA (4 mM)
• DTT (1 M)
• Dilution Buffer
Description:
Activation of ICE-family
proteases/caspases initiates apoptosis in mammalian cells. The
FLICE/Caspase-8 Colorimetric Assay Kit provides a simple and convenient
means for assaying the activity of caspases that recognize the sequence
IETD. The assay is based on spectrophotometric detection of the
chromophore p-nitroanilide (pNA) after cleavage from the labeled
substrate IETD-pNA. The pNA light emission can be quantified using a
spectrophotometer or a microtiter plate reader at 400- or 405 nm.
Storage Conditions:
-20°C
Shipping Conditions:
gel pack
Caspase-8 Assay 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).
• After thawing, store the Cell Lysis Buffer and dilution Buffer at 4°C.
• Protect IETD-pNA from light.
B. 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 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 (optional).
7. Dilute 100-200 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. Add 5 µl of the 4 mM IETD-pNA substrate (200 µM final conc.). 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 cuvette (Sigma), or dilute sample to 1 ml with Dilution Buffer and using regular cuvette (note: Dilution of the samples proportionally decreases the reading).
You may also perform the entire assay in a 96-well plate.
Fold-increase in FLICE activity can be determined by comparing the results of treated samples 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 FLICE activity.
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