235418 Caspase-3 Inhibitor Screening Assay Kit

235418
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Fluorometric or Colorimetric
      Description
      OverviewA convenient assay kit useful for screening caspase-3 inhibitors measuring the protease activity of caspase-3 and other caspase-3-like activities. Cleavage is monitored colorimetrically by measuring the increase in absorbance at 405 nm.
      Catalogue Number235418
      Brand Family Calbiochem®
      Materials Required but Not Delivered Plate reader capable of measuring A405 to ≥3-decimal accuracy.
      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.
      References
      ReferencesThornberry, N.A., and Lazebnik, Y. 1998. Science 281, 1312.
      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.
      Darmon, A.J., et al. 1995. Nature 377, 446.
      Lazebnik, Y.A., et al. 1995. Proc. Natl. Acad. Sci. USA 92, 9042.
      Nicholson, D.W., et al. 1995. Nature 376, 37.
      Tewari, M., et al. 1995. Cell 81, 801.
      Fernandes-Alnemri, 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 (Cat. No. 235417), a DEVD-pNA Colorimetric Substrate, DEVD-AMC Fluorometric Substrate, Calibration Standard I (p-Nitroaniline), Calibration Standard II (AMC), an Inhibitor, Assay Buffer, ½-volume 96-Well Plate, and a user protocol.
      Applications
      Biological Information
      Assay time2 h
      Sample TypeRecombinant or purified caspase-3
      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 of kit store entire kit Contents (except 1/2 volume well plate, store at room temperature) 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 (Cat. No. 235417), a DEVD-pNA Colorimetric Substrate, DEVD-AMC Fluorometric Substrate, Calibration Standard I (p-Nitroaniline), Calibration Standard II (AMC), an Inhibitor, Assay Buffer, ½-volume 96-Well Plate, and a user protocol.
      Specifications

      Documentation

      Caspase-3 Inhibitor Screening Assay Kit Certificates of Analysis

      TitleLot Number
      235418

      References

      Reference overview
      Thornberry, N.A., and Lazebnik, Y. 1998. Science 281, 1312.
      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.
      Darmon, A.J., et al. 1995. Nature 377, 446.
      Lazebnik, Y.A., et al. 1995. Proc. Natl. Acad. Sci. USA 92, 9042.
      Nicholson, D.W., et al. 1995. Nature 376, 37.
      Tewari, M., et al. 1995. Cell 81, 801.
      Fernandes-Alnemri, T., et al. 1994. J. Biol. Chem. 269, 30761.

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      User Protocol

      Revision20-September-2010 RFH
      Form96 Tests
      Format96-well plate
      Detection methodFluorometric or Colorimetric
      StorageUpon arrival of kit store entire kit Contents (except 1/2 volume well plate, store at room temperature) at -70°C.
      BackgroundCaspase-3 (also known as CPP32, apopain, and Yama), a member of the interleukin-1b converting enzyme (ICE) family of cysteine proteases, is composed of 17 and 12 kDa subunits derived from 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. Targets of caspase-3 cleavage include poly(ADP-ribose) polymerase (PARP), nuclear lamins, and others.
      Principles of the assayThe Calbiochem® Caspase-3 Inhibitor Screening Assay Kit is designed to measure the protease activity of caspase-3. It contains both a colorimetric substrate (DEVD-pNA) and a fluorogenic substrate (DEVD-AMC). Cleavage of the p-nitroanilide (pNA) from the colorimetric substrate results in an increased absorption at 405 nm. The fluorescent assay is based on the cleavage of 7-amino-4-methylcoumarin (AMC) from the C-terminus of the fluorogenic substrate, which is measured by the increase in fluorescence intensity at 460 nm. The assays are performed in a convenient 96-well plate format. The kit is useful for screening inhibitors of caspase-3 that may have a potential therapeutic value. A caspase-3 inhibitor (Ac-DEVD-CHO) is also included as a prototypic control inhibitor. The DEVD amino acid sequence is derived from the caspase-3 cleavage site in PARP.
      Materials provided• Assay Buffer (Kit Component No. KP1201): 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. KP1202): Supplied as 5000 units (100 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. KP1203): Supplied as 1 ml of 2 mM Ac-DEVD-pNA (1.3 mg/ml) in assay buffer. M.W. 638.6.
      • Caspase-3 Inhibitor I (Ac-DEVD-CHO) (Kit Component No. KP1205): Supplied as 50 µl of 100 µM Ac-DEVD-CHO (50 µg/ml) in DMSO. M.W. 502.5.
      • 1/2 volume 96-Well Plate (Kit Component No. KP1206): 1 each
      • Calibration Standard I (Kit Component No. KP1207): Supplied as 1 ml of 50 µM pNA in assay buffer. M.W. 138.1.
      • Caspase Substrate II Fluorometric (Ac-DEVD-AMC) (Kit Component No. KP1208): Supplied as 1 ml of 0.3 mM Ac-DEVD-AMC (200 µg/ml) in assay buffer.
      • Calibration Standard II (Kit Component No. KP1209): Supplied as 1 ml of 30 µM AMC in assay buffer.
      • Vial Holder (Kit Component No. KP1210): 1 each.
      Materials Required but not provided Plate reader capable of measuring A405 to ≥3-decimal accuracy.
      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.
      Detailed protocolNote on Storage: For greater stability, store all components, except the plate, at -70°C. The Caspase-3 enzyme must be handled carefully in order to retain maximal enzymatic activity. Defrost quickly in a bath set at room temperature or by rubbing between fingers, then immediately store in an ice bath. Any unused enzyme should be quickly refrozen by placing at -70°C. The enzyme is stable to approximately 4 freeze/thaw cycles. To minimize the number of freeze/thaw cycles, aliquot caspase-3 into separate vials and store at -70°C.

      Note: It is important to determine the plate reader conversion factor before beginning the assay. See step 7 of the Data Analysis for guidelines.

      Assay Procedure:

      1. Thaw all kit components and hold in ice bath until use. All kit components are stable for several h in an ice bath.
      2. Briefly warm Caspase-3 Inhibitor I to room temperature. In a separate microcentrifuge tube, dilute Caspase-3 Inhibitor I (1:200) in Assay Buffer by adding 1 µl inhibitor to 200 µl Assay Buffer.
      3. You will need 25 µl of Caspase-3 per well. Dilute (1:50) in Assay Buffer to required quantity needed for the experiment. For example, add 5 µl Caspase-3 to 245 µl assay buffer in a separate tube.
      4. Dilute Caspase Substrate I, colorimetric (Ac-DEVD-pNA) or Caspase Substrate II, Fluorometric (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 diluted substrate to assay temperature, e.g. 30°C.
      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. Allow plate to equilibrate to assay temperature, e.g., 30°C.
      7. Add 25 µl Caspase-3 (diluted in step 3 above) to the control, inhibitor and test sample wells. Final amount of Caspase-3 will be 50 U per well. DO NOT ADD CASPASE-3 TO BLANKS.
      8. Add 25 µl Caspase-3 Inhibitor I (diluted in step 2 above) to the appropriate wells. Final inhibitor concentration = 0.1 µM.
      9. Add desired volume of test sample to 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 Caspase-3 Substrate II, Fluorometric (pre-equilibrated to assay temperature). Final substrate concentration = 200 µM for pNA substrate and 30 µM for AMC substrate.
      12. Continuously read absorbance at 405 nm for the pNA substrate or fluorescence for the AMC substrate,in a plate-reader. Record data at 1 min intervals for 10 to 60 min. 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. 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 versus time for each sample.

      Figure 1: Example of plot of A405 versus time



      2. Determine the length of the initial time period over which the reaction rate is linear, and there is sufficient change in absorbance to obtain an accurate slope. Typically, points from 1 to 15 min are sufficient.
      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.
      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. To find the percent remaining activity of the inhibitor:

      % remaining inhibitor activity = (inhibitor slope / control slope) x 100

      7. To find the activity of the samples expressed as pmol substrate/min using chromogenic substrate (pNA chromophore), determine plate reader conversion factor:
      a) Add 100 µl (assay volume) pNA calibration standard [50 µM p-nitroaniline (pNA) in the assay buffer] to 2 wells of the 1/2 volume plate. Typically, 100 µl of the 50 µM standard, in a 1/2 volume well, produces an A405 of about 0.3. Alternatively, build a pNA standard curve and use the slope (µM/OD) as the conversion factor.
      b) Determine the average A405 using 100 µl (assay volume) assay buffer as a blank.
      c) Calculate the conversion factor.

      Conversion factor (µM/A405) = 50 µM/average A405 from step b

      d) Calculate the activity as pmol/min:

      activity (pmol/min) = slope (O.D./min) x conversion factor (µM/OD x assay vol (µl)

      Figure 2: Sample Activity Calculation



      8. To find the activity of the samples expressed as pmol substrate/min using fluorogenic substrate (AMC fluorophore):
      a) Determine microplate reader conversion factor for AMC fluorophore. 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 µM/RFU 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. The slope of the standard curve can then be used as the µM/RFU conversion factor.

      b) Calculate the activity as pmol/min:
      activity (pmol/min) = slope (RFU/sec) x 60sec/min x conversion factor(µM/RFU) x assay vol (µl)
      Example data

      Figure 3: Example of Inhibitor Data

      Control slope = 3.4E-03 ΔA/min Inhibitor slope = 1.0E-04 ΔA/min % inhibitor activity remaining = (1E-04/3.4E-03) x 100 = 2.9%

      Figure 4: Example graph for Km and Vmax determination (Eadie-Hofstee

      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.

      User Protocols

      Title
      User Protocol