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235400 Caspase-3 Substrate I, Colorimetric - CAS 1177131-27-9 - Calbiochem

235400
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Overview

Replacement Information

Key Spec Table

Products

Catalogue NumberPackaging Qty/Pack
235400-5MG Plastic ampoule 5 mg
Description
OverviewColorimetric substrate for caspase-3 (Km = 9.7 µM) and related cysteine proteases. Sequence is based on the P1-P4 tetrapeptide cleavage site of poly(ADP-ribose) polymerase (PARP) and includes Asp216. Caspase-3 is the protease responsible for the cleavage of PARP. Also a substrate for caspase-6, caspase-7, caspase-8, and caspase-10. Cleavage of pNA is monitored colorimetrically at ~405 nm.
Catalogue Number235400
Brand Family Calbiochem®
SynonymsAc-DEVD-pNA
References
ReferencesCardone, M.H., et al. 1998. Science 282, 1318.
Thornberry, N.A., and Lazebnik, Y. 1998. Science 281, 1312.
Nicholson, D.W., et al. 1995. Nature 376, 37.
Lazebnik, Y.A., et al. 1994. Nature 371, 346.
Product Information
CAS number1177131-27-9
ATP CompetitiveN
FormLyophilized
Hill FormulaC₂₆H₃₄N₆O₁₃
Chemical formulaC₂₆H₃₄N₆O₁₃
ReversibleN
Structure formula ImageStructure formula Image
Quality LevelMQ100
Applications
Biological Information
Primary TargetCaspas-3
Primary Target IC<sub>50</sub>Km = 9.7 µM as colorimetric substrate for caspase-3
Purity≥95% by HPLC
Physicochemical Information
Cell permeableN
Peptide SequenceAc-Asp-Glu-Val-Asp-pNA
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Ambient Temperature Only
Toxicity Standard Handling
Storage -20°C
Protect from Light Protect from light
Protect from Moisture Protect from moisture
Do not freeze Ok to freeze
Special InstructionsFollowing reconstitution, aliquot and freeze (-20°C). Stock solutions are stable for up to 6 months at -20°C.
Packaging Information
Transport Information
Supplemental Information
Specifications

Documentation

Caspase-3 Substrate I, Colorimetric - CAS 1177131-27-9 - Calbiochem SDS

Title

Safety Data Sheet (SDS) 

Caspase-3 Substrate I, Colorimetric - CAS 1177131-27-9 - Calbiochem Certificates of Analysis

TitleLot Number
235400

References

Reference overview
Cardone, M.H., et al. 1998. Science 282, 1318.
Thornberry, N.A., and Lazebnik, Y. 1998. Science 281, 1312.
Nicholson, D.W., et al. 1995. Nature 376, 37.
Lazebnik, Y.A., et al. 1994. Nature 371, 346.
Data Sheet

Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

Revision31-March-2011 RFH
SynonymsAc-DEVD-pNA
DescriptionColorimetric substrate for CPP32/Apopain/Yama (Km = 9.7 µM) and related cysteine proteases. Sequence is based on the P1-P4 tetrapeptide cleavage site of poly (ADP-ribose) polymerase (PARP) and includes Asp216. CPP32 is the protease responsible for the cleavage of PARP. Can be used with Pro-caspase-3 in in vitro assays to measure caspase-9 activity, since caspase-9 activates pro-caspase-3. Cleavage of the substrate can be monitored at 405 nm (ε = 9160 cm-1M-1).
FormLyophilized
Recommended reaction conditions
This is a general protocol designed for use with a plate reader. The amount of cell extract, substrate, inhibitor, and p-nitroaniline (pNA) used is for an assay volume of 100 µl. The procedure may be scaled up for use in a spectrophotometer.

Materials Required:
1. Caspase-3 Substrate I, Colorimetric (Cat. No. 235400)
2. Caspase-3 Inhibitor I (Cat. No. 235420)
3. Cell Lysis Buffer: 50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA
4. Assay Buffer: 50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 0.1 mM EDTA and 10% glycerol
5. Phosphate Buffered Saline (PBS)
6. Plate and a plate reader

Recommended Additional Materials:
1. Purified Caspase-3
2. p-nitroaniline (pNA)

Preparation of Cell Extracts:
1. Induce apoptosis in cells by using an apoptosis inducing agent. Appropriate controls should include untreated cells, cells treated with an inactive analog of the apoptosis inducer (if available), or a "time-zero" sample from the apoptosis induction time course.

A sufficient number of cells are needed for duplication of the assay, for use as an inhibitor control, and for determination of protein concentration. In general, the number of cells described below will give an adequate protein concentration for the plate assay, but keep in mind that differences in cell size, volume, and protein concentration may necessitate increased plating densities. If the cell density at lysis is 2 x 107 cells/ml, the protein concentration will be about 1-3 mg/ml and a 10 µl assay sample will contain 10-30 µg of protein. Depending on the cell line, a minimum of 106 cells in 50 µl lysis buffer is required.

2. Count cells and harvest by centrifugation. Wash cells once with PBS. If the cells have been treated with a potential caspase inhibitor, it may be necessary to wash more thoroughly to prevent inhibition of the assay.
3. Resuspend the cells to the desired concentration using ice-cold lysis buffer. Incubate for 5 min on ice.
4. Centrifuge at 10,000 x g for 10 min at 4°C.
5. Collect the supernatant (cytosolic extract) and hold on ice until use. Extracts can be flash frozen in an acetone/ethanol bath and stored at -70°C for later use.

Assay Procedure:
1. Prepare a 100 mM solution of Caspase-3 Substrate I in DMSO. Prepare a 1:50 dilution (2 mM) of the stock solution in Assay Buffer. Aliquot and freeze (-20°C) the remaining DMSO stock solution.
2. Prepare a 100 mM solution of Caspase-3 Inhibitor I in DMSO. Prepare a 1:1000 dilution (100 µM) in assay buffer and then prepare a working 5X (500 nM) stock solution by further diluting 5 µl with 1 ml of Assay Buffer. Aliquot and freeze (-20°C) the remaining DMSO stock solution.
3. Add the appropriate amount of Assay Buffer to each well of the plate see table below.
Blank and cell extract samples are essential for determining cellular activity. To measure non-specific hydrolysis of Caspase-3 Substrate I, preparation of an inhibitor-treated cell extract is recommended. Using a positive-control sample which includes purified Caspase-3 is also recommended. See table:

Table 1: Assay Mixture Examples

* One unit is defined as the amount of enzyme that will cleave 1.0 pmol of the substrate per minute at 30°C, pH 7.4.


4. Allow the plate to equilibrate to 37°C. The assay may also be performed at room temperature, but the rate of substrate cleavage will decrease by ~30%.
5. Add 10 µl of cell extract to the appropriate wells. Do not add cell extract to the blank (control) wells.
6. Add 20 µl Caspase-3 Inhibitor I (final concentration 100 nM) or add test inhibitor to the appropriate wells.
7. Incubate the plate at the assay temperature for 10 min (or as desired) to allow enzyme/inhibitor interaction.
8. Initiate the reaction by adding 10 µl of Caspase-3 Substrate I, which has been pre-equilibrated to the assay temperature. The final concentration will be 200 µM.
9. Measure the absorbance at 405 nm in a plate reader. Record data at 1-10 min intervals for 30-120 min.

Qualitative Measurement of Caspase-3 Activity:
1. Plot data as A405 nm versus time for each sample.
2. For each sample, determine the initial time period over which the plot of absorbance (Ab) versus time remains linear, and determine if there is a sufficient change in absorbance to obtain an accurate slope. Saturation will occur using the initial substrate concentration (200 µM Caspase-3 Substrate I). For most samples, the rate of substrate cleavage will remain constant for 2 h or more. Since highly active samples may reduce the substrate to sub-saturating levels during the course of the experiment, data from the early linear portion of the curve should be used to calculate the slope. Using a suitable linear regression program, obtain the slope of the line. Average the slopes of replicate samples.
3. If the blank has a significant slope, adjust the sample data accordingly.

Quantitative Measurement of Caspase-3 Activity:
1. To determine the plate reader conversion factor, prepare a 50 µM stock solution of pNA standard in Assay Buffer. Add 100 µl to 2 wells.
2. Determine the average A405 nm using 100 µl of Assay Buffer as a blank.
3. Calculate the conversion factor. This calculation is based on the concentration of pNA in the calibration standard (50 µM). The extinction coefficient for pNA in the assay buffer is 10,000 M-1cm-1.

Conversion factor (mM/Ab) = 50 µM/Average A405 nm

4. Calculate the activity as pmol substrate hydrolyzed/min:

Activity (pmol/min) = slope (Ab/min) x conversion factor (µM/Ab) x assay volume (µl)
CAS number1177131-27-9
Chemical formulaC₂₆H₃₄N₆O₁₃
Peptide SequenceAc-Asp-Glu-Val-Asp-pNA
Structure formulaStructure formula
Purity≥95% by HPLC
SolubilityDMSO (60 mg/ml)
Storage -20°C
Protect from moisture
Protect from light
Do Not Freeze Ok to freeze
Special InstructionsFollowing reconstitution, aliquot and freeze (-20°C). Stock solutions are stable for up to 6 months at -20°C.
Toxicity Standard Handling
ReferencesCardone, M.H., et al. 1998. Science 282, 1318.
Thornberry, N.A., and Lazebnik, Y. 1998. Science 281, 1312.
Nicholson, D.W., et al. 1995. Nature 376, 37.
Lazebnik, Y.A., et al. 1994. Nature 371, 346.