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CBA023 InnoZyme™ Cathepsin L Activity Kit, Fluorogenic

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CBA023
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Overview

Replacement Information

Key Spec Table

Detection Methods
Fluorometric

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      Description
      OverviewA highly sensitive and selective fluorogenic assay for the detection of Cathepsin L from human and mouse samples. Cathepsin L is detected with the fluorogenic substrate Z-Phe-Arg-AMC (Exc. max: 360 nm; Emi. max: 460 nm). Interference from Cathepsin B is eliminated by the incorporation of CA 074, a specific cathepsin B inhibitor. This kit is also useful for screening cathepsin L inhibitors.
      Catalogue NumberCBA023
      Brand Family Calbiochem®
      Application Data
      Calibration curve generated using the AMC Calibration Standard included in the InnoZyme™ Cathepsin L Activity Kit. The assay was carried out as specified in the Cathepsin L Inhibitor Screening protocol.
      Materials Required but Not Delivered• Purified Cathepsin L (for inhibitor screening only)
      • Protein assay (for biological samples)
      • Precision pipettors or multi-channel precision pipettor; use only pipettors that are carefully calibrated to their target volume
      • 37°C incubator/shaker
      • Microplate reader capable of measuring fluorescence at wavelengths 360-380 nm for excitation and 440-460 nm for emission
      • Ice bucket
      References
      ReferencesBohne, S., et al. 2004. J. Pathol.203, 528.
      Goulet, B., et al. 2004. Mol. Cell 14, 207.
      Hook, V., et al. 2004. Biol. Chem. 385, 473.
      Potts, W., et al. 2004. Int. J. Exp. Pathol. 85, 85.
      Rousselet, N., et al. 2004. Cancer Res. 64, 146.
      Schedel, J., et al. 2004. Gene Ther. 11, 1040.
      Troy, A.M. et al. 2004. Eur. J. Cancer 40, 1610.
      Corella, R., and Casey, S.F. 2003. Biotech. Histochem. 78, 101.
      Ohashi, K., et al. 2003. Biochim. Biophys. Acta 1649, 30.
      Friedrich, B., et al. 1999. Eur. J. Cancer 35, 138.
      Cresswell P., 1998. Science 280, 394.
      Yasuma, T., et al. 1998. J. Med. Chem. 41, 4301.
      Kirschke, H. et al. 1982. Biochem. J. 201, 367.
      Product Information
      Detection methodFluorometric
      Form96 Tests
      Format96-well plate
      Kit containsCathepsin L Substrate, AMC Calibration Standard, Cathepsin L Control, Cathepsin L Activator, CA 074, Cathepsin L Inhibitor, Assay Buffer, Sample Buffer, Diluent, Microwell Plate, Plate Sealer, and a user protocol.
      Positive controlCathepsin L
      Quality LevelMQ100
      Applications
      Biological Information
      Assay range1.56-100 ng/ml
      Assay time1.5 - 2 h
      Sample TypeCell lysates, tissues, or purified cathepsin L
      Physicochemical Information
      Sensitivity1.2 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® InnoZyme™ Cathepsin L Activity Kit, Fluorogenic is suited for measuring Cathepsin L activity using purified preparations, cell lysates, and tissue extracts, and for screening Cathepsin L inhibitors. This assay does not measure Cathepsin B activity.
      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 Multiple storage requirements
      Storage ConditionsUpon arrival store the Cathepsin L Control at -70°C; store all other unopened components at -20°C. Upon initial use of the kit, the components may be stored for up to 3 months under the following conditions:

      Store at 4°C: Assay Buffer, Sample Buffer Diluent, 96-Well Plate, and Plate Sealers
      Store at -20°C: AMC Calibration Standard, CA 074 Inhibitor, Cathepsin L Inhibitor Substrate, and Reduction Reagent
      Store at -70°C: Cathepsin L Control: dispense into aliquots upon initial use; avoid freeze/thaw cycles
      Protect from Light Protect from light
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCathepsin L Substrate, AMC Calibration Standard, Cathepsin L Control, Cathepsin L Activator, CA 074, Cathepsin L Inhibitor, Assay Buffer, Sample Buffer, Diluent, Microwell Plate, Plate Sealer, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA023-1KITCN 04055977220889

      Documentation

      InnoZyme™ Cathepsin L Activity Kit, Fluorogenic SDS

      Title

      Safety Data Sheet (SDS) 

      InnoZyme™ Cathepsin L Activity Kit, Fluorogenic Certificates of Analysis

      TitleLot Number
      CBA023

      References

      Reference overview
      Bohne, S., et al. 2004. J. Pathol.203, 528.
      Goulet, B., et al. 2004. Mol. Cell 14, 207.
      Hook, V., et al. 2004. Biol. Chem. 385, 473.
      Potts, W., et al. 2004. Int. J. Exp. Pathol. 85, 85.
      Rousselet, N., et al. 2004. Cancer Res. 64, 146.
      Schedel, J., et al. 2004. Gene Ther. 11, 1040.
      Troy, A.M. et al. 2004. Eur. J. Cancer 40, 1610.
      Corella, R., and Casey, S.F. 2003. Biotech. Histochem. 78, 101.
      Ohashi, K., et al. 2003. Biochim. Biophys. Acta 1649, 30.
      Friedrich, B., et al. 1999. Eur. J. Cancer 35, 138.
      Cresswell P., 1998. Science 280, 394.
      Yasuma, T., et al. 1998. J. Med. Chem. 41, 4301.
      Kirschke, H. et al. 1982. Biochem. J. 201, 367.

      Citations

      Title
    • Moin U Fareed, et al. (2006) Treatment of rats with calpain inhibitors prevents sepsis-induced muscle proteolysis independent of atrogin-1/MAFbx and MuRF1 expression. American Journal of Physiology Regulatory, Integrative and Comparative Physiology 290, R1589-R1597.
      		•
      User Protocol

      Revision28-November-2016 JSW
      Form96 Tests
      Format96-well plate
      Detection methodFluorometric
      StorageUpon arrival store the Cathepsin L Control at -70°C; store all other unopened components at -20°C. Upon initial use of the kit, the components may be stored for up to 3 months under the following conditions:

      Store at 4°C: Assay Buffer, Sample Buffer Diluent, 96-Well Plate, and Plate Sealers
      Store at -20°C: AMC Calibration Standard, CA 074 Inhibitor, Cathepsin L Inhibitor Substrate, and Reduction Reagent
      Store at -70°C: Cathepsin L Control: dispense into aliquots upon initial use; avoid freeze/thaw cycles
      Intended useThe Calbiochem® InnoZyme™ Cathepsin L Activity Kit, Fluorogenic is suited for measuring Cathepsin L activity using purified preparations, cell lysates, and tissue extracts, and for screening Cathepsin L inhibitors. This assay does not measure Cathepsin B activity.
      BackgroundCathepsin L is a lysosomal endoproteinase that belongs to the papain family of cysteine proteinases. It plays a major role in antigen processing, tumor metastasis, bone resorption, and turnover of intracellular and secreted regulatory proteins. It degrades many protein substrates, some of which include collagen, IL-8 precursor, neurotransmitter precursor, pro-enkephalin, and immunoglobulin light chain-associated (AL) amyloid deposits. It is also responsible for cleaving other proenzymes, like Cathepsin D, resulting in enzyme activation. Increased Cathepsin L activity is linked to some cancers including prostate and colorectal cancers and melanoma, where it promotes degradation of extracellular matrix and basement membrane that lead to tumor metastasis. It also plays a role in the pathology of degenerative cartilage and bone disorders. Cathepsin L homozygous knockout mice are reported to have a significant reduction in bone volume. Additionally, Cathepsin L-deficient mice exhibit distinct defects in MHC class II processing in thymic cortical epithelial cells. Goulet, et al, 2004, report that a truncated, alternative active form of the enzyme is involved in the regulation of cell cycle progression via translocation to the nucleus, where it fully activates the transcription factor, CDP/Cux. Cathepsin B is a closely related proteinase that generally resides in the same cellular compartments as Cathepsin L and is known to cleave the same or similar substrates. As a result of this close association, it is also present in Cathepsin L-containing samples, making it difficult to distinguish Cathepsin L activity from Cathepsin B and vice versa. The InnoZyme™ Cathepsin L Activity Kit, Fluorogenic is designed to assess the activity of Cathepsin L without interference from Cathepsin B.
      Principles of the assayThe InnoZyme™ Cathepsin L Activity Kit, Fluorogenic utilizes a fluorogenic peptide substrate, Z-Phe-Arg-7-amido-4-methylcoumarin, HCl, in an enzyme cleavage assay to measure the activity of Cathepsin L in a 96-Well Plate. The Substrate can also be cleaved by Cathepsin B, whose activity in crude samples must first be inhibited by the addition of CA 074 Inhibitor, a specific and irreversible Cathepsin B inhibitor. Cathepsin L activity in biological samples is measured in the presence of CA 074 Inhibitor alone and in the presence of CA 074 Inhibitor and the irreversible Cathepsin L Inhibitor (Z-FY(t-Bu)-DMK). Cleavage of the Substrate by Cathepsin L releases AMC and the fluorescence is measured at 360 nm excitation and 460 nm emission. The readout is reported as relative fluorescent units (RFU). RFU of the samples are determined by subtracting the RFU as measured in the presence of CA 074 Inhibitor and Cathepsin L Inhibitor from the RFU measured in the presence of CA 074 Inhibitor alone. The unit activity of Cathepsin L can be determined by generating a calibration curve using the AMC Calibration Standard. The assay is optimized for use with a 96-Well Plate, but can be adapted to a cuvette format.

      When using the assay to assess the activity of purified Cathepsin L, the addition of CA 074 is not necessary. Cathepsin L activity in purified preparations is measured in the presence and absence of Cathepsin L Inhibitor. For unknown inhibitor screening, activity is also measured in the presence and absence of unknown inhibitor(s). RFU of the samples are determined by subtracting the RFU as measured in the presence of Cathepsin L Inhibitor from the RFU measured in the absence of Cathepsin L Inhibitor.

      Interference from other lysosomal, cysteine proteinases is minimal; <1 % for human cathepsin B, H, and S, and <2 % for human cathepsin K.
      Materials provided• Substrate (Kit Component No. JA7950): 1 vial, 10 mM Z-Phe-Arg-7-amido-4-methylcoumarin, HCl in DMSO, supplied as a 100X solution
      • AMC Calibration Standard (Kit Component No. JA7742): 1 vial, 2 mM AMC in DMSO, supplied as a 100X solution
      • Cathepsin L Control (Kit Component No. JA7951): 1 vial, Cathepsin L, Human liver, supplied a 10 µg/ml
      • CA 074 Inhibitor (Kit Component No. JA7952): 1 vial, in DMSO, supplied as a 100X solution
      • Cathepsin L Inhibitor (Kit Component No. JA7953): 1 vial, 1 mM Z-FY(t-Bu)-DMK in DMSO, supplied as a 100X solution
      • Assay Buffer (Kit Component No. JA7954): 1 vial,
      • Sample Buffer (Kit Component No. JA7955): 1 bottle
      • Diluent (Kit Component No. JA7956): 1 bottle
      • Reduction Reagent (Kit Component No. JA7665): 1 vial, TCEP supplied as a 125X solution
      • 96-Well Plate (Kit Component No. JA7666): 1 plate, 96 wells, supplied as six 16-well strips
      • Plate Sealer (Kit Component No. JB155): 2 plate sealers, reusable
      Materials Required but not provided• Purified Cathepsin L (for inhibitor screening only)
      • Protein assay (for biological samples)
      • Precision pipettors or multi-channel precision pipettor; use only pipettors that are carefully calibrated to their target volume
      • 37°C incubator/shaker
      • Microplate reader capable of measuring fluorescence at wavelengths 360-380 nm for excitation and 440-460 nm for emission
      • Ice bucket
      Preparation• Biological Samples (e.g. crude extracts): Dilute samples, as necessary, using Sample Buffer. Typically, 10-200 µg protein/50 µl is recommended. • Purified Cathepsin L Sample: Dilute purified Cathepsin L, as necessary, using Sample Buffer. Typically, 10-100 ng/ml of enzyme is recommended.
      Reagent preparationAll reagents necessary to perform the assay are supplied with this kit. Bring all reagents, except the Cathepsin L Control (see below), to room temperature prior to use. • AMC Calibration Standard: The AMC Calibration Standard is supplied as a 2 mM stock solution. The recommended concentration range for generating a calibration curve is 0.156-20 µM. Prepare serial dilutions of the Calibration AMC Standard as indicated in the table below. Vortex each dilution prior to transfer, taking care to change the pipet tip for each dilution. • Cathepsin L Control: Prepare desired number of Cathepsin L Control samples by diluting the Cathepsin L Control (10 µg/ml stock) with Sample Buffer, within the range of 1.56-100 ng/ml. Typically 0.078-5 ng/50 µl is recommended. Thaw and prepare diluted Cathepsin L Control just prior to use. Dispense the remaining Cathepsin L Control into aliquots and freeze at -70°C. • Activation Buffer: For measuring Cathepsin L activity in biological samples, prepare enough Activation Buffer for 50 µl/well. Dilute Reduction Reagent 1:125 and CA 074 Inhibitor 1:100 in Assay Buffer. Example: to prepare 1 ml of Activation Buffer, enough for a 16-well strip, add 10 µl CA 074 Inhibitor and 8 µl Reduction Reagent to 982 µl Assay Buffer. Note: to prepare Activation Buffer for Cathepsin L inhibitor screening do not add CA 074 Inhibitor to the Activation Buffer; add only Reduction Reagent. • Cathepsin L Inhibitor Working Solution (1X): Dilute Cathepsin L Inhibitor 1:100 with Diluent. Example: to prepare 1 ml Cathepsin L Inhibitor Working Solution, enough for a 16-well strip, add 10 µl Cathepsin L Inhibitor to 990 µl Diluent. • Substrate Working Solution (1X): Dilute the Substrate (10 mM) 1:100 with Diluent. Example: to prepare 1 ml Substrate Working Solution, enough for a 16-well strip, add 10 µl Substrate to 990 µl Diluent. • Test Inhibitor: Dilute any Test Inhibitors to the desired concentration with Diluent in a final volume of 50 µl.
      Detailed protocolMeasuring Cathepsin L Activity in Biological Samples

      1. Prepare all reagents as indicated in the Reagent Preparation section above. Remove the desired number of strips from the 96-Well Plate, return the unused strips to the foil pouch, and seal the entire edge of the pouch. Store unused strips at 4°C.
      2. Add 50 µl Activation Buffer to each well.
      3. Prepare the Calibration AMC Standard reactions by adding 50 µl of each AMC Calibration Standard dilution to designated wells. Prepare Cathepsin L Control reactions by adding 50 µl of each diluted Cathepsin L Control to designated wells. Prepare Biological Sample reactions by adding 50 µl of each sample to designated wells. Prepare Blank reactions by adding 50 µl Sample Buffer to designated wells.
      4. Cover the plate with the Plate Sealer and incubate for 15 min at room temperature with gentle shaking to allow for activation of Cathepsin L by the Reducing Reagent in the Activation Buffer and inhibition of any Cathepsin B by CA 074 Inhibitor.
      5. Add 50 µl Cathepsin L Inhibitor Working Solution (1X) to designated wells. Add 50 µl Diluent to the remaining wells.
      6. Cover the plate with the Plate Sealer and incubate for 15 min at room temperature with gentle shaking.
      7. Add 50 µl Substrate Working Solution (1X) to each well.
      8. Cover the plate with the Plate Sealer and incubate for 1-2 h at 37°C.
      Note: seal the plate tightly to prevent evaporation. Any changes in the reaction volume can influence the fluorescence reading.
      9. Read and record the fluorescence as measured at an excitation of 360-380 nm and emission of 460-480 nm in a fluorescence plate reader.

      Cathepsin L Inhibitor Screening


      1. Prepare all reagents as indicated in the Reagent Preparation section above. Remove the desired number of strips from the 96-Well Plate, return the unused strips to the foil pouch, and seal the entire edge of the pouch. Store unused strips at 4°C.
      2. Add 50 µl Activation Buffer to each well.
      3. Prepare the Calibration AMC Standard reactions by adding 50 µl of each AMC Calibration Standard dilution to designated wells. Prepare purified Cathepsin L reactions by adding 50 µl of purified Cathepsin L to designated wells. Prepare Blank reactions by adding 50 µl Sample Buffer to designated wells. If desired, prepare Cathepsin L Control reactions by adding 50 µl of each diluted Cathepsin L Control to designated wells.
      4. Cover the plate with the Plate Sealer and incubate for 3 min. at room temperature with gentle shaking to allow for activation of Cathepsin L by the Reducing Reagent in the Activation Buffer.
      5. Add 50 µl Cathepsin L Inhibitor Working Solution (1X) to designated wells. Add 50 µl Test Inhibitor to designated wells. Add Diluent to the remaining wells.
      6. Cover the plate with the Plate Sealer and incubate for 4-5 min. at room temperature with gentle shaking.
      7. Add 50 µl Substrate Working Solution (1X) to each well.
      8. Cover the plate with the Plate Sealer and incubate for 1-2 h at 37°C with gentle shaking.
      Note: seal the plate tightly to prevent evaporation. Any changes in the reaction volume can influence the fluorescence reading.
      9. Read and record the fluorescence as measured at an excitation of 360-380 nm and emission of 460-480 nm in a fluorescence plate reader.
      CalculationsAssay with AMC Calibration: It is recommended that the data be handled by a software package utilizing a linear regression curve-fitting program. If a software package is not available, construct a standard curve by plotting the mean fluorescence in relative fluorescence units (RFU), for each standard on the Y-axis versus the corresponding concentration of AMC on the X-axis and draw a best-fit curve through the points on the graph. A. Final RFU for Cathepsin L Sample with CA074 = (Crude (initial) RFU value for Cathepsin L Sample with CA074) - (RFU of Blank) B. Final RFU for Cathepsin L Sample with CA074 and cathepsin L inhibitor = (Crude RFU for Cathepsin L Sample with CA 074 and cathepsin L inhibitor) - (RFU of Blank) C. Corrected RFU for Cathepsin L sample = Final RFU from A - Final RFU from B The Cathepsin L activity of unknown samples, using the final RFU, can be interpolated from the standard curve and displayed as an amount of free AMC released/mg protein in a specified time period. Assay without AMC Calibration: All biological samples should be assayed in duplicate and in parallel with two different conditions, in the presence of both CA 074 Inhibitor and Cathepsin L Inhibitor and in the presence of CA 074 Inhibitor alone. The initial Cathepsin L activity, displayed as RFU, is calculated by subtracting the RFU value for CA 074 Inhibitor + Cathepsin L Inhibitor from the RFU value for CA 074 Inhibitor only. The final Cathepsin L activity in each sample is displayed in RFU and is derived by subtracting the value of the blank from the initial RFU and then calculating the mean fluorescence value for duplicate samples. If the samples were diluted, the final Cathepsin L activity, as calculated above, is multiplied by the dilution factor to obtain the final Cathepsin L activity.
      Standard curve
      Example data
      Sensitivity1.2 ng/ml
      Sensitivity NotesThe minimum detectable activity (sensitivity) is 1.2 ng/ml and is defined as two standard deviations above the mean RFU of 16 replicates containing all reaction components except enzyme. The corresponding concentration was calculated from a control curve set up in duplicates.
      Assay Range1.56-100 ng/ml
      Assay Range NotesThe working range of the assay, as determined using highly purified Cathepsin L, is between 1.56-100 ng/ml or 0.078-5 ng/assay.
      Plate configuration
      Registered TrademarksCalbiochem® and His•Tag® are registered trademarks of EMD Chemicals, Inc.
      CytoBuster™, Interactive Pathways™ and InnoZyme™ are trademarks of EMD Chemicals, Inc.