BioVision rev.
10/11 For research use only
Caspase-3/CPP32 Colorimetric Assay Kit
(Catalog #K106-25, -100, -200, -400; Store kit at –20°C)
I. Introduction:
0.52
Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. 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.
II. Kit Contents:
Components K106-25 K106-100 K106-200 K106-400 Part Number
25 assays 100 assays 200 assays 400 assays 0.04
Cell Lysis Buffer 25 ml 100 ml 100 ml 100 ml K106-XX(X)-1
2X Reaction Buffer 2 ml 4 x 2 ml 16 ml 32 ml K106-XX(X)-2
DEVD-pNA (4 mM) 125 µl 0.5 ml 2 x 0.5 ml 2 x 1 ml K106-XX(X)-3 Uninduced Induced
DTT (1 M) 100 µl 0.4 ml 0.4 ml 0.4 ml K106-XX(X)-4
Dilution Buffer 25 ml 100 ml 200 ml 400 ml K106-XX(X)-5
Induction of Caspase-3 Activity by Anti-Fas Antibody in Jurkat –T
III. Caspase-3 Assay Protocol: Cells Using Caspase-3 Colorimteric Assay Kit K106-25
A. General Considerations
IV. Storage and Stability:
• 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 Store kit at –20 ° C (Store Lysis Buffer, Reaction Buffer, and Dilution Buffer at 4 ° C after
of 1.0 M DTT stock per 1 ml of 2X Reaction Buffer). opening). All reagents are stable for at least 6 months under proper storage conditions.
• Protect DEVD-pNA from light.
B. Assay Procedure VI. Related Products:
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture Apoptosis Detection Kits & Reagents
without induction. • Annexin V Kits & Bulk Reagents
2. Count cells and pellet 1-5 x 106 cells. • Caspase Assay Kits & Reagents
3. Resuspend cells in 50 µl of chilled Cell Lysis Buffer and incubate cells on ice for 10 • Mitochondrial Apoptosis Kits & Reagents
minutes. • Nuclear Apoptosis Kits & Reagents
4. Centrifuge for 1 min in a microcentrifuge (10,000 x g). • Apoptosis Inducers and Set
5. Transfer supernatant (cytosolic extract) to a fresh tube and put on ice for immediate
Cell Fractionation System
assay or aliquot and store at –80°C for future use.
• Mitochondria/Cytosol Fractionation Kit
6. Assay protein concentration.
7. Dilute 50-200 µg protein to 50 µl Cell Lysis Buffer for each assay. • Nuclear/Cytosol Fractionation Kit
8. Add 50 µl of 2X Reaction Buffer (containing 10 mM DTT) to each sample. • Membrane Protein Extraction Kit
9. Add 5 µl of the 4 mM DEVD-pNA substrate (200 µM final conc.) and incubate at 37°C for • Cytosol/Particulate Rapid Separation Kit
1-2 hour. • Mammalian Cell Extraction Kit
10. Read samples at 400- or 405-nm in a microtiter plate reader, or spectrophotometer • FractionPREP Fractionation System
using a 100-µl micro quartz cuvette (Sigma), or dilute sample to 1 ml with Dilution Buffer Cell Proliferation & Senescence
and using regular cuvette (note: Dilution of the samples proportionally decreases the • Quick Cell Proliferation Assay Kit
reading). Fold-increase in CPP32 activity can be determined by comparing these results • Senescence Detection Kit
with the level of the uninduced control.
• High Throughput Apoptosis/Cell Viability Assay Kits
Note: Background reading from cell lysates and buffers should be subtracted from the
• LDH-Cytotoxicity Assay Kit
readings of both induced and the uninduced samples before calculating fold increase in
CPP32 activity • Bioluminescence Cytotoxicity Assay Kit
• Live/Dead Cell Staining Kit
BioVision Research Products Tel: 650-428-0236 • Fax: 650-428-0336
980 Linda Vista Avenue, Mountain View, CA 94043 USA www.biovision.com •
[email protected] Page 1
BioVision rev. 10/11 For research use only
GENERAL TROUBLESHOOTING GUIDE FOR CASPASE COLORIMETRIC AND FLUOROMETRIC KITS:
Problems Cause Solution
Assay not working • Cells did not lyse completely • Resuspend the cell pellet in the lysis buffer and incubate as described in the datasheet
• Experiment was not performed at optimal time after • Perform a time-course induction experiment for apoptosis
apoptosis induction
• Plate read at incorrect wavelength • Check the wavelength listed in the datasheet and the filter settings of the instrument
• Old DTT used • Always use freshly thawed DTT in the cell lysis buffer
High Background • Increased amount of cell lysate used • Refer to datasheet and use the suggested cell number to prepare lysates
• Increased amounts of components added due to incorrect • Use calibrated pipettes
pipetting
• Incubation of cell samples for extended periods • Refer to datasheet and incubate for exact times
• Use of expired kit or improperly stored reagents • Always check the expiry date and store the individual components appropriately
• Contaminated cells • Check for bacteria/ yeast/ mycoplasma contamination
Lower signal levels • Cells did not initiate apoptosis / assay not done at optimal • Determine the time-point for initiation of apoptosis after induction (time-course experiment)
timepoint after induction of apoptosis
• Very few cells used for analysis • Refer to datasheet for appropriate cells number
• Use of samples stored for a long time • Use fresh samples or aliquot and store and use within one month for the assay
• Incorrect setting of the equipment used to read samples • Refer to datasheet and use the recommended filter setting
• Allowing the reagents to sit for extended times on ice • Always thaw and prepare fresh reaction mix before use
Samples with erratic readings • Uneven number of cells seeded in the wells • Seed only equal number of uniformly suspended healthy cells (correct passage number)
• Samples prepared in a different buffer • Use the cell lysis buffer provided in the kit
• Adherent cells dislodged and lost at the time of experiment • Perform experiment gently and in duplicates/triplicates; apoptotic cells may become floaters
• Cell/ tissue samples were not completely homogenized • Use Dounce homogenizer (increase the number of strokes); observe efficiency of lysis under
microscope
• Samples used after multiple freeze-thaw cycles • Aliquot and freeze samples, if needed to use multiple times
• Presence of interfering substance in the sample • Trouble-shoot as needed
• Use of old or inappropriately stored samples • Use fresh samples or store at correct temperatures until use
Unanticipated results • Measured at incorrect wavelength • Check the equipment and the filter setting
• Cell samples contain interfering substances • Trouble shoot if it interferes with the kit (run proper controls)
General issues • Improperly thawed components • Thaw all components completely and mix gently before use
• Incorrect incubation times or temperatures • Refer to datasheet & verify the correct incubation times and temperatures
• Incorrect volumes used • Use calibrated pipettes and aliquot correctly
• Air bubbles formed in the well/tube • Pipette gently against the wall of the well/tubes or spin down the plate
• Substituting reagents from older kits/ lots • Use fresh components from the same kit
• Use of a different 96-well plate • Fluorescence: Black plates; Absorbance: Clear plates
Note# The most probable cause is listed under each section. Causes may overlap with other sections.
BioVision Research Products Tel: 650-428-0236 • Fax: 650-428-0336
980 Linda Vista Avenue, Mountain View, CA 94043 USA www.biovision.com •
[email protected] Page 2
