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235419 Caspase-3 Cellular Activity Assay Kit

Caspase-3 Cellular Activity Assay Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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235419
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Overview

Replacement Information

Key Spec Table

Species ReactivityDetection Methods
A Broad Range Of SpeciesFluorometric or colorimetric

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      Description
      OverviewA convenient assay kit useful for measuring the caspase-3 and caspase-3-like activities in cell extracts using either a colorimetric or fluorometric substrate. Provided in a convenient 96-well format with all reagents necessary for preparing cell extracts, measuring caspase activity and calibrating the assay. In addition, caspase-3 is included for use as a positive control, for testing cell extracts for endogenous inhibitors, or for comparing effects of exogenous inhibitors on cellular activity versus the activity of the purified enzyme.
      Catalogue Number235419
      Brand Family Calbiochem®
      Materials Required but Not Delivered• plate reader capable of measuring A405 to ≥3-decimal accuracy or fluorescent plate reader capable of reading at an excitation of wavelength of 360 nm and an emission wavelength of 460 nm.
      • Pipettor or any multi-channel pipettor capable of pipetting 10-100 µl accurately. Note: dilution of reagents can be made to increase the minimal volume to >10 µl.
      • Ice bucket to keep reagents cold until use.
      • Apoptosis-induced cultured cells.
      • Centrifuge
      • Phosphate buffered saline (PBS).
      References
      ReferencesThornberry, N.A., and Lazebnik, Y. 1998. Science 281, 1312.
      Faleiro, L., et al. 1997. EMBO J. 16, 2271.
      Kothakota, S., et al. 1997. Science 278, 294.
      Nicholson, D.W. 1996. Nat. Biotechnol. 14, 297.
      Schlegel, J., et al. 1996. J. Biol. Chem. 271, 1841.
      Srinivasula, S.M., et al. 1996. Proc. Natl. Acad. Sci. USA 93, 14486.
      Nicholson, D.W., et al. 1995. Nature 376, 37.
      Tewari, M., et al. 1995. Cell 81, 801.
      Fernandes-Alnemi, T., et al. 1994. J.Biol. Chem. 269, 30761.
      Product Information
      Detection methodFluorometric or colorimetric
      Form96 Tests
      Format96-well plate
      Kit containsHuman Recombinant Caspase-3, Cell Lysis Buffer, a DEVD-pNA Colorimetric Substrate, Ac-DEVD-AMC Fluorometric Substrate, Calibration Standard (p-Nitroaniline), AMC Calibration Standard, an Inhibitor, Assay Buffer, one half-volume 96-Well Plate, and a user protocol.
      Quality LevelMQ100
      Applications
      Biological Information
      Assay time2.5-3 h
      Sample TypeCell extracts
      Species Reactivity
      • A Broad Range Of Species
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 23/24/25-63-36/37/38-33

      Toxic by inhalation, in contact with skin and if swallowed.
      Possible risk of harm to the unborn child.
      Irritating to eyes, respiratory system and skin.
      Danger of cumulative effects.
      S PhraseS: 36/37/39-45-26-23

      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Do not breathe fumes.
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage ≤ -70°C
      Storage ConditionsUpon arrival store the 1/2 volume plate at room temperature and the remaining components of the kit at -70°C.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsHuman Recombinant Caspase-3, Cell Lysis Buffer, a DEVD-pNA Colorimetric Substrate, Ac-DEVD-AMC Fluorometric Substrate, Calibration Standard (p-Nitroaniline), AMC Calibration Standard, an Inhibitor, Assay Buffer, one half-volume 96-Well Plate, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      235419-1KIT 07790788048884

      Documentation

      Caspase-3 Cellular Activity Assay Kit SDS

      Title

      Safety Data Sheet (SDS) 

      Caspase-3 Cellular Activity Assay Kit Certificates of Analysis

      TitleLot Number
      235419

      References

      Reference overview
      Thornberry, N.A., and Lazebnik, Y. 1998. Science 281, 1312.
      Faleiro, L., et al. 1997. EMBO J. 16, 2271.
      Kothakota, S., et al. 1997. Science 278, 294.
      Nicholson, D.W. 1996. Nat. Biotechnol. 14, 297.
      Schlegel, J., et al. 1996. J. Biol. Chem. 271, 1841.
      Srinivasula, S.M., et al. 1996. Proc. Natl. Acad. Sci. USA 93, 14486.
      Nicholson, D.W., et al. 1995. Nature 376, 37.
      Tewari, M., et al. 1995. Cell 81, 801.
      Fernandes-Alnemi, T., et al. 1994. J.Biol. Chem. 269, 30761.

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      User Protocol

      Revision02-June-2010 JSW
      Form96 Tests
      Format96-well plate
      Detection methodFluorometric or colorimetric
      Speciesa broad range of species
      StorageUpon arrival store the 1/2 volume plate at room temperature and the remaining components of the kit at -70°C.
      BackgroundCaspase-3 (also known as CPP32, apopain, and Yama), a member of the interleukin-1β converting enzyme (ICE) family of cysteine proteases, is composed of 17 and 12 kDa subunits derived from a common proenzyme, pro-caspase-3. It is activated during apoptotic signaling events by upstream proteases including caspase-6, caspase-8 (FLICE), and cytotoxic T-cell-derived granzyme B. Caspase-3 is one of the principal caspases found in apoptotic cells. Targets of caspase-3 cleavage include poly(ADP-ribose) polymerase (PARP), nuclear lamins, gelsolin, and others. Caspase-3 is a potential therapeutic target.
      Principles of the assayThe Calbiochem® Caspase-3 Cellular Activity Assay Kit is designed to measure caspase-3 and caspase-3-like activity in cell extracts. It is provided in a convenient 96-well format with all reagents necessary for preparing cell extracts, measuring caspase activity, and calibrating the assay. For your convenience, we have included caspase-3 enzyme for use as a positive control and for testing cell extracts for endogenous inhibitors. If the kit is used to screen agents that affect cellular caspase activity, the purified enzyme provides a ready means to check for direct effect of these agents on caspase-3.
      Materials provided• Cell Lysis Buffer (Kit Component No. KP1301-30ML): Supplied as 30 ml of 50 mM HEPES, 5 mM DTT, 0.1 mM EDTA, 0.1% CHAPS, pH 7.4.
      • Assay Buffer (Kit Component No. KP1302-20ML): Supplied as 20 ml of 100 mM NaCl, 50 mM HEPES, 10 mM DTT, 1 mM EDTA, 10% glycerol, 0.1% CHAPS, pH 7.4.
      • Caspase-3, Human, Recombinant (Kit Component No. KP1303-300U): Supplied as 300 units (10 units/µl) in assay buffer. One unit is the amount of enzyme that will release 1 pmol of pNA from 200 µM DEVD-pNA per min at 30°C. AVOID FREEZE/THAW CYCLES.
      • Caspase Substrate I, Colorimetric (Ac-DEVD-pNA) (Kit Component No. KP1304-1ML): Supplied as 1 ml of 2 mM (1.3 mg/ml) in assay buffer. M.W. 638.6.
      • p-Nitroaniline (Kit Component No. KP1305-1ML): Supplied as 1 ml of 50 µM solution in assay buffer. A405nm = 0.525 cm⁻¹. M.W. 138.1.
      • Caspase-3 Inhibitor I (Ac-DEVD-CHO) (Kit Component No. KP1306-50UL): Supplied as 50 µl of 100 µM (0.05 mg/ml) in DMSO. M.W. 502.5.
      • 1/2 Volume Plate (Kit Component No. KP1307-1EA): 1 each
      • Fluorometric substrate (Ac-DEVD-AMC) (Kit Component No. KP1308-1ML): Supplied as 1 ml of 0.3 mM solution in assay buffer. M.W. 676.
      • 7-Amino-4-methylcoumarin (AMC) Calibration Standard (Kit Component No. KP1309-1ML): Supplied as 1 ml of 30 µM solution in assay buffer. M.W. 175.
      Materials Required but not provided• plate reader capable of measuring A405 to ≥3-decimal accuracy or fluorescent plate reader capable of reading at an excitation of wavelength of 360 nm and an emission wavelength of 460 nm.
      • Pipettor or any multi-channel pipettor capable of pipetting 10-100 µl accurately. Note: dilution of reagents can be made to increase the minimal volume to >10 µl.
      • Ice bucket to keep reagents cold until use.
      • Apoptosis-induced cultured cells.
      • Centrifuge
      • Phosphate buffered saline (PBS).
      PreparationPreparation of Cell Extracts 1. Grow cell cultures and induce apoptosis as desired using an appropriate agent. Appropriate controls may include untreated cells, cells treated with an inactive chemical analog of the apoptosis inducer or simply the "time-zero" sample from an apoptosis induction time course. The number of cells required for an experiment must be determined by the user. A sufficient quantity is needed to assay caspase-3 activity, plus additional material to determine protein concentration (as needed). As a guide, here is an example of data obtained with U937 cells: • Protein concentration = 1 - 3 mg/ml [cell density (at lysis) = ~2 x 107 cells/ml] • 10 µl assay sample = 10-30 µg protein (or ~2 x 105 cells) • DEVD-pNA cleavage assays: 10 ml samples; 37°C; A405 after 30 min. - control cells = 0.004, apoptotic cells = 0.22. It is desirable to have enough extract to perform duplicate assays, with and without an inhibitor-treated control and determine protein concentration. Thus, for U937 cells, a minimum of 106 cells in 50 µl for each test condition is suggested. Other cell types may have other requirements. 2. Count cells and harvest by centrifugation (e.g.: 1000 x g, 4°C, 10 min). Wash cells once with phosphate buffered saline (PBS). If the cells have been treated with a reagent that may interfere with the subsequent caspase assay (e.g. a potential caspase inhibitor), it may be desirable to wash cells more extensively prior to lysis. 3. Resuspend cells to desired concentration (e.g.: 2 x 107/ml) with ice-cold Cell Lysis Buffer. Incubate for 5 min on ice. If cell lysis is incomplete, additional detergent may be added to the Cell Lysis Buffer to assist membrane solubilization/destabilization. Addition of TWEEN®-20 detergent (Cat. No. 655205 or 655206), NP-40 (Cat. No. 492015 or 492017), or TRITON® X-100 detergent (Cat. No. 648462 or 648463) to a final concentration of 0.1% is compatible with the subsequent caspase assay. 4. Centrifuge at 10,000 x g, 10 min at 4°C. 5. Save supernatant (cytosol) and hold on ice bath until use. Alternatively, the extracts may be quickly frozen and stored at -70°C for later use. For highest stability, store all reagents at -70°C. Store the plate at room temperature. Caspase-3 must be handled with particular care at room temperature in order to retain its maximal enzymatic activity. Thaw it quickly in a water bath or by rubbing between fingers, then immediately store on an ice bath. Any unused enzyme should be quickly refrozen at -70°C. To minimize degradation and loss of enzyme activity, aliquot Caspase-3 into several tubes and store at -70°C.
      Detailed protocolDetermining the Plate Reader Conversion Factor

      Note: It is important to determine the plate reader conversion factor before beginning the assay.
      To find the activity of the samples expressed as pmol substrate/min, determine plate reader conversion factor:
      a) Add 100 µl (assay volume) substrate standard [50 µM p-nitroaniline (pNA) in the assay buffer] to 2 wells of the 1/2 volume plate.
      b) Determine the average A405 using 100 µl (assay volume) assay buffer as a blank.
      c) Calculate the conversion factor. This calculation is based on the concentration of p-nitroaniline in the calibration standard (50 µM). The extinction coefficient for p-nitroaniline in assay buffer is 10,500 M-1cm-1.
      Conversion factor (µM/ A405) = 50 µM/average A405 from step b
      d) Calculate the activity as pmol/min:
      activity (pmol/min) = slope (DA/min) x conversion factor x assay vol (µl)
      Assay volume is 100 µl for the standard assay (Table 1). NOTE: If a different volume is used, be sure to perform steps a) to d) using the actual assay volume.

      Figure 1: Sample Activity Calculation


      1. Thaw all kit components and hold on ice bath until use. All kit components are stable for several h in an ice bath. If using frozen cell extracts, handle in the same manner as caspase-3.
      2. Dilute Caspase-3 Inhibitor (Ac-DEVD-CHO; briefly warm to room temperature to thaw) 1:200 in Assay Buffer by adding 1 µl inhibitor to 200 µl Assay Buffer in a separate tube. This solution contains 0.5 µM inhibitor and will serve as a 5X stock for preparing inhibitor-treated controls.
      3. Dilute the Caspase-3 Substrate I, Colorimetric (Ac-DEVD-pNA) or Fluorometric Substrate (Ac-DEVD-AMC) in Assay Buffer to 2X the desired final concentration. For example, dilute Ac-DEVD-pNA to 400 µM (final 200 µM) or Ac-DEVD-AMC to 60 µM (final 30 µM). Equilibrate the dilution to assay temperature (e.g. 37°C).
      4. You will need 15 µl (30 U) of Caspase-3 per well. Dilute (1:5) in Assay Buffer to required quantity needed for the experiment. For example, add 10 µl Caspase-3 to 40 µl assay buffer to make 3 samples.
      5. Add Assay Buffer to each well of the 1/2 volume plate as required. The final volume of each reaction will be 100 µl. Table 1 lists examples with the reagent volumes needed for several experimental conditions.
      6. The "Blank" and "Cell Extract" samples are essential for determining cellular activity. Two additional controls are highly recommended: 1) Inhibitor-Treated Cell Extract to measure nonspecific hydrolysis of Caspase-3 Substrate I, Colorimetric or Fluorometric Substrate (see Figure 5, "+ Caspase-3 Inhibitor I"); 2) Purified Caspase-3 which provides a positive control and standard with which to compare cellular activities (see Figure 2).
      Note: The two "Test Sample" assays listed in Table 1 illustrate a format that may be useful for inhibitor screening/drug discovery work. Endogenous inhibitory activity in cell extracts may be assessed by comparing the rate obtained in the "Cell Extract/Purified Caspase-3" assay with the rate obtained separately with the cell extract or purified enzyme.
      7. Allow the plate to equilibrate to assay temperature (e.g.: 37°C). Note: The assays illustrated in Figs. 2-6 were performed at 37°C. Similar data were obtained at 25°C; however, rates of Caspase-3 Substrate I, Colorimetric cleavage were ~2/3 of those obtained with the same samples at 37°C.
      8. Add 10 µl of cell extracts or 15 µl caspase-3 (diluted in step 3 above) to the appropriate wells (see Table 1). DO NOT ADD CELL EXTRACTS OR CASPASE-3 TO BLANKS.
      9. Add test sample and/or 20 µl Caspase-3 Inhibitor I (diluted in step 2 above; final concentration = 0.1 µM) to the appropriate wells (see Table 1).
      10. Incubate plate at assay temperature for 10 min (or as desired) to allow inhibitor/enzyme interaction.
      11. Start reaction by adding 50 µl Caspase-3 Substrate I, Colorimetric or 50 µl Fluorometric Substrate (pre-equilibrated to assay temperature). Final substrate concentration = 200 µM for the colorimetric substrate and 30 µM for the fluorometric substrate.
      12. Read absorbance at 405 nm for the colorimetric substrate or the fluorescence at 360 nm (excitation)/460 nm (emission) for the fluorometric substrate. Record data at 5-10 min intervals for 30 to 120 min (see Figure 2). NOTE: Retain plate for future use of unused wells.
      13. Analyze the data (see below).

      Table 1: Assay Mixture Examples

      *Test sample refers to an experimental inhibitor/activator. Dissolve/dilute inhibitor into assay buffer and add to appropriate wells at desired volume "Y". Adjust volume "X" to bring the total volume to 100 µl.
      Calculations1. Plot data as A405 or arbitrary fluorescence units (AFU) versus time for each sample. 2. For each sample, determine the length of the initial time period over which the plot of absorbance (or AFU) vs. time remains linear, and there is sufficient change in absorbance (or change in AFU) to obtain an accurate slope. The initial substrate concentration (200 µM DEVD-pNA) is saturating. For many samples the rate of DEVD-pNA cleavage will remain constant for at least 2 h. Highly active samples, however, can reduce the substrate concentration to sub-saturating levels in substantially less time. In such cases, choose data from the earlier, linear portion of the time course for use in the slope calculation of Step 3. (For an example, see the "Etop.-4" data in Fig. 2). Since the AMC substrate is used at a sub-saturating concentration (30 µM), extra care must be taken in choosing data that lies within the linear portion of the reaction progress curve. 3. Obtain the slope of the line, fitted to the linear portion of the data, using an appropriate linear regression program. 4. Average the slopes of replicate samples. DATA ANAYLSIS (in terms of substrate conversion) 5. If the blank has a significant slope, subtract this number from all samples. Under normal conditions this will not be necessary since the slope will be nearly zero. 6. Specific Activity Calculations a) To normalize activities with respect to cell number, divide the values calculated in step 6 by the number of cells suspended in 10 µl of lysis buffer. (see Experimental Methods, steps 1-3): Specific Activity (pmol/min/number of cells) = activity (pmol/min)/number of cells per well b) To calculate specific activities with respect to total protein, determine the protein content of each cell extract. Divide activities by the protein content of the corresponding sample (see Figures 2 and 3): Specific Activity = pmol/min/mg protein Note: The cell lysis buffer is compatible with Coomassie dye binding (Bradford) and bicinchoninic acid (BCA) based protein assays under appropriate conditions. For the BCA assay, cell extract samples must be diluted ≥10-fold in water. Increased backgrounds may be exhibited with the BCA assays due to the increased DTT concentration. Cell extracts diluted >10-fold with water are considered compatible. Be sure to use cell lysis buffer in the standards and maintain equal amounts in all samples. Note regarding AMC calibration standard: The exact AMC concentration range that will be useful for preparing a standard curve will vary depending on the fluorimeter model, the gain setting, and the exact excitation and emission wavelengths used. The AMC standard, as provided (30 µM), may yield off-scale readings in some cases. We recommend diluting some of the standard to a relatively low concentration with Assay Buffer (0.5 or 1.0 µM) and then measuring the fluorescence of 100 µl. The estimate of AFU/µM obtained with this measurement, together with the observed range of values obtained in the enzyme assays, can then be used to plan an appropriate series of dilutions for a standard curve.
      Assay characteristics and examples

      Figure 2: Cleavage of DEVD-pNA by Cell Extracts and Purified Caspase-3.

      U937 cells (a human histocytic lymphoma) were induced to undergo apoptosis by treatment with 50 µM Etoposide (Cat. No. 341205). Extracts were prepared as described in Experimental Methods. Etop.-1, -2 etc. indicate h of etoposide treatment. "Control" cell extract was prepared from untreated cells. The "Pur. Csp-3" sample contained 30 U of Caspase-3 (Cat. No. 235417). Assay temperature: 37°C.


      Figure 3: Cleavage of DEVD-AMC by Cell Extracts

      Jurkat cells were treated with 2 µg/ml human TRAIL ligand for 90 min to induce apoptosis. Cell extracts were prepared and assayed according to the protocol outlined above. 10 µl Jurkat cell extract, 30 µM DEVD-AMC, 37°C.


      Figure 4: Induction of Caspase-3 Activity in Etoposide-Treated U937 Cells

      Caspase-3 Substrate I, Colorimetric (DEVD-pNA) cleavage rates were calculated from the slopes of the best-fit lines in Fig. 2. Protein content of each sample was determined with a BCA assay. After 3 h of treatment, ~50% of cells were morphologically apoptotic (2 or more membrane "blebs" per cell).


      Figure 5: Induction of Caspase-3 Activity in TNF-a-Treated U937 Cells

      U937 cells were treated with 2 ng/ml tumor necrosis factor -α (TNF-α) plus 0.5 µg/ml Cycloheximide (Cat. No. 239764). After 1 h of treatment, ~50% of cells were morphologically apoptotic.


      Figure 6: DEVD-CHO Inhibition of Caspase-3-Like Activity in Cell Extracts

      Extracts were prepared from untreated (control), etoposide, and TNF-α treated U937 cells as described under Experimental Methods and Figs. 2-4. Prior to assay of DEVD-pNA cleavage, extracts were preincubated for 10 min at 37°C with or without 0.1 µM Caspase-3 Inhibitor I (see "Caspase-3 Assay," step 5).
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      TRITON® is a registered trademark of Rohm and Haas Company
      TWEEN® is a registered trademark of ICI Americas Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.