QIA120 Calpain Activity Assay Kit, Fluorogenic

QIA120
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Fluorometric

      Pricing & Availability

      Catalog Number AvailabilityPackaging Qty/Pack Price Quantity
      QIA120-1KIT
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          Glass bottle 1 kit
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          Description
          OverviewCalpains are Ca2+-dependent proteinases, which are involved in normal cellular processes including cell proliferation, apoptosis, differentiation, and cell migration. Requires a fluorimeter or microplate reader capable of measuring fluorescence at wavelengths of Excitation max: ~360 nm and Emission max: ~460 nm.
          Catalogue NumberQIA120
          Brand Family Calbiochem®
          Application Data
          Typical standard curve generated by using dilutions of the AMC Standard as indicated in the Detailed Protocol.
          Materials Required but Not Delivered Pipetman or multi-channel pipetman: use only pipettes that have been carefully calibrated to their target volume
          Microplate reader capable of measuring fluorescence at an excitation wavelength of ~360-380 nm and an emission wavelength of ~440-460 nm
          Ice bucket to keep reagents cold until use
          Phosphate-buffered saline (PBS) for preparation of cell lysates
          References
          ReferencesDainese, E., et al. 2002. J. Biol. Chem. 277, 40296.
          Glading, A., et al. 2000. J. Biol. Chem. 275, 23908.
          Ishikara, I., et al. 2000. Neurosci. Lett. 279, 97.
          Nakagawa, T. and Yuan, J. 2000. J. Cell Biol. 150, 887.
          Pariat, M., et al. 2000. Biochem. J. 345, 129.
          Pink, J.J., et al. 2000.Exp. Cell Res. 255, 144.
          Wang, K.K. 2000. Trends Neurosci. 23, 20.
          Reddy, R.K., et al. 1999. J. Biol. Chem. 274, 28476.
          Leist, M., et al. 1998. Mol. Pharmacol. 54, 789.
          Melloni, E., et al. 1998. J. Biol. Chem. 273, 12827.
          Villa, P.G., et al. 1998. J. Cell Sci. 111, 713.
          Wood, D.E., et al. 1998. Oncogene 17, 1069.
          Kubbutat, M.H. and Vousden, K.H. 1997. Mol. Cell Biol. 17, 460.
          Molinari, M. and Carafoli, E. 1997. J. Membr. Biol. 156, 1.
          Sorimachi, H., et al. 1997. Biochem. J. 328, 721.
          Squier, M.K. and Cohen, J.J. 1997. J. Immunol. 158, 3690.
          Aoki, K., et al. 1986. FEBS Lett. 205, 313.
          Ohno, S., et al. 1986. Nucleic Acid Res. 14, 5559.
          Product Information
          Detection methodFluorometric
          Form96 Tests
          Format96-well plate
          Kit containsCalibration Standard, Positive Control Calpain 1, Substrate, Activation Buffer, Inhibition Buffer, Reduction Agent, Assay Buffer, Cell Lysis Buffer, 96-Well Plate, Plate Sealer, and a user protocol.
          Positive controlCalpain 1
          Applications
          Biological Information
          Assay time1.5 h
          Sample TypeCell lysates, plasma, serum
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Storage and Shipping Information
          Ship Code Multiple Storage Conditions
          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 Control Human Calpain 1 at -70°C and the remaining components at -20°C. With the exception of the Control Human Calpain 1, all components, once opened, can be stored at 4°C for up to 1 month.
          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 containsCalibration Standard, Positive Control Calpain 1, Substrate, Activation Buffer, Inhibition Buffer, Reduction Agent, Assay Buffer, Cell Lysis Buffer, 96-Well Plate, Plate Sealer, and a user protocol.
          Specifications

          Documentation

          Calpain Activity Assay Kit, Fluorogenic SDS

          Title

          Safety Data Sheet (SDS) 

          Calpain Activity Assay Kit, Fluorogenic Certificates of Analysis

          TitleLot Number
          QIA120

          References

          Reference overview
          Dainese, E., et al. 2002. J. Biol. Chem. 277, 40296.
          Glading, A., et al. 2000. J. Biol. Chem. 275, 23908.
          Ishikara, I., et al. 2000. Neurosci. Lett. 279, 97.
          Nakagawa, T. and Yuan, J. 2000. J. Cell Biol. 150, 887.
          Pariat, M., et al. 2000. Biochem. J. 345, 129.
          Pink, J.J., et al. 2000.Exp. Cell Res. 255, 144.
          Wang, K.K. 2000. Trends Neurosci. 23, 20.
          Reddy, R.K., et al. 1999. J. Biol. Chem. 274, 28476.
          Leist, M., et al. 1998. Mol. Pharmacol. 54, 789.
          Melloni, E., et al. 1998. J. Biol. Chem. 273, 12827.
          Villa, P.G., et al. 1998. J. Cell Sci. 111, 713.
          Wood, D.E., et al. 1998. Oncogene 17, 1069.
          Kubbutat, M.H. and Vousden, K.H. 1997. Mol. Cell Biol. 17, 460.
          Molinari, M. and Carafoli, E. 1997. J. Membr. Biol. 156, 1.
          Sorimachi, H., et al. 1997. Biochem. J. 328, 721.
          Squier, M.K. and Cohen, J.J. 1997. J. Immunol. 158, 3690.
          Aoki, K., et al. 1986. FEBS Lett. 205, 313.
          Ohno, S., et al. 1986. Nucleic Acid Res. 14, 5559.

          Brochure

          Title
          The Active Siteル Volume 4, Number 1

          Citations

          Title
        • Afsaneh Abdolzade-Bavil, et al. (2004) Convenient and versatile subcellular extraction procedure, that facilitates classical protein expression profiling and functional protein analysis. Proteomics 4, 1397-1405.
        • Ashish K. Sharma and Baerbel Rohrer. (2004) Calcium-induced Calpain Mediates Apoptosis via Caspase-3 in a Mouse Photoreceptor Cell Line. Journal of Biological Chemistry 279, 35564-35572.
        • Gloria Bonuccelli, et al. (2003) Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins. American Journal of Pathology 163, 1663-1675.
        • User Protocol

          Revision14-December-2009 RFH
          Form96 Tests
          Format96-well plate
          Detection methodFluorometric
          StorageUpon arrival, store the Control Human Calpain 1 at -70°C and the remaining components at -20°C. With the exception of the Control Human Calpain 1, all components, once opened, can be stored at 4°C for up to 1 month.
          BackgroundCalpains are cytosolic Ca2+-dependent cysteine proteases that are widely distributed in all mammalian cells. Calpains 1 and 2 are activated in vitro by micromolar (10-50 µM) or millimolar (0.2-0.35 mM) levels of Ca2+, respectively. In vivo, calpain 1 appears to be activated at physiological Ca2+ concentrations of 100-300 nM, suggesting the involvement of other activators. Calpain 1 is heterodimeric protein comprised of a large 78-80 kDa catalytic subunit encoded by capn1, and a 28-30 kDa regulatory subunit encoded by capn4. Calpains may play a central role in the execution of apoptosis either upstream or downstream of caspases in, e.g. glucocorticoid-treated and irradiated thymocytes, neuronal cells exposed to various stresses, or MCF-7 breast cancer cells treated with b-lapachone. Interestingly, caspases and calpains share several substrates (the endogenous calpain inhibitor calpastatin, fodrins, focal adhesive kinase, calmodulin-dependent kinases, actin, vimentin, and keratins), suggesting that these enzymes may carry out cell death in a partially indistinguishable manner. Calpains can also cleave other potentially interesting apoptosis-related proteins including caspase 12, Bax, Bcl-XL, GRP94, c-Fos, and p53.
          Principles of the assayThe Calbiochem® Calpain Activity Kit, Fluorogenic is suitable for the quantitative determination of calpain-1 and calpain-2 activity in 96-well plates. The assay utilizes the synthetic calpain substrate, Suc-LLVY-AMC, in the presence of calpain-containing samples, Ca2+ and the reducing agent TCEP. AMC is released upon cleavage with calpain and is measured fluorometrically at an excitation wavelength of ~360-380 nm and an emission wavelength of ~440-460 nm. Calpain can be quantitated using an AMC calibration curve or can be displayed in relative fluorescence units (RFU). The kit includes highly purified native human calpain 1 as a positive control, which should be included in every assay.

          The analysis of calpain activity in biological samples requires that the assay be performed with specific activation and inhibition buffers. The calpain activity is determined by subtracting the activity obtained using the inhibition buffer from the activity detected with the activation buffer. The inhibition buffer contains the calcium chelator BAPTA, which completely blocks calpain activity. For customer convenience, CytoBuster™ Protein Extraction Reagent (Cat.No.71009) is included for preparation of cell lysates.
          Materials provided• AMC Standard (Kit Component No. JA7660-100UL): 1 vial, 100 µl, 1 mM 7-Amino-4-methylcoumarin (AMC) in DMSO supplied as a 100-fold concentrated solution.
          • Control Human Calpain 1 (Kit Component No. JA7662-20UL): 2 vials, 20 µl each of highly purified Calpain 1 from human erythrocytes
          • Substrate (Kit Component No. JA7661-60UL): 1 vial, 60 µl Suc Leu-Leu-Val-Tyr-AMC supplied as a 100-fold concentrated solution
          • Activation Buffer (Kit Component No. JA7663-20ML): 1 bottle, 20 ml buffer containing Ca2+; requires the addition of TCEP reducing agent just prior to use
          • Inhibition Buffer (Kit Component No. JA7664-10ML): 1 bottle, 10 ml buffer containing BAPTA, supplied as a ready-to-use solution
          • Reduction Reagent (Kit Component No. JA7665-100UL): 1 vial, 100 µl 0.5 M TCEP provided as a 250-fold concentrated solution
          • Assay Buffer (Kit Component No. JA7667-25ML): 1 bottle, 25 ml assay buffer for sample, control, AMC standard, and substrate dilutions
          • Cell Lysis Buffer (Kit Component No. 71009-10ML): 1 bottle, 10 ml CytoBuster™ Protein Extraction Reagent
          • 96-well Plate (Kit Component No. JA7666-1EA): 1 plate
          Materials Required but not provided Pipetman or multi-channel pipetman: use only pipettes that have been carefully calibrated to their target volume
          Microplate reader capable of measuring fluorescence at an excitation wavelength of ~360-380 nm and an emission wavelength of ~440-460 nm
          Ice bucket to keep reagents cold until use
          Phosphate-buffered saline (PBS) for preparation of cell lysates
          Preparation1. Cell lysates: a. Wash cell pellet with ice-cold PBS. b. Add 500-1000 µl Lysis Buffer (approximately 1 ml per 1 x 107 cells) and incubate on ice for 30 min. c. Vortex and centrifuge the lysate at 14,000 x g in a pre-cooled tabletop microcentrifuge. d. Transfer the supernatant to a fresh tube immediately and discard the pellet. e. Dilute the lysate at least 1:10 before determining the protein concentration using a BCA protein assay. 2. Dilute samples with Assay Buffer if necessary. Typically, cell lysates with a protein concentration >3 mg/ml should be diluted 5-fold. If the measured RFU exceeds 6000 the sample should be diluted further.
          Reagent preparationNote: All reagents necessary to perform the assay are supplied with this kit. Warm all components (except the Control Human Calpain-1) to 15-25°C (room temperature) before use; store the Control Human Calpain-1 on ice until use. For best results prepare dilutions of reagents only as needed. • Diluted Standards: The AMC standard is supplied as a 1 mM stock solution (100-fold concentrated solution). To prepare a calibration curve use serial dilutions of the standard ranging from a concentration of 0.625-10 µM Example: Label 6 eppendorf tubes. Pipet 495 µM Assay Buffer into the first tube and 200 µl into the remaining tubes. Add 5 µl AMC Standard to the first tube. Vortex and transfer 200 µl from this tube to the next tube. Continue serial dilutions by transferring 200 µl to consecutive tubes up to tube 5. The tube labeled 6 will contain Assay Buffer only (Blank). • Diluted Substrate: Dilute Substrate 100-fold with Assay Buffer. Example: to prepare enough Substrate for 6 strips dilute 30 µl Substrate with 2.97 ml Assay Buffer. • Activation Buffer, Final: The Reduction Reagent (TCEP) must be added to the Activation Buffer just prior to use. Example: to prepare Activation Buffer, Final add 20 µl Reduction Reagent 5 ml Activation Buffer. • Control Human Calpain-1: Dilute the Control Human Calpain-1 with Assay Buffer as indicated on the vial label. The Control Human Calpain-1 should be stored at -70°C to maintain activity. Once thawed and diluted it should be used immediately; diluted enzyme can be stored on ice for a few h.
          Detailed protocolExcept for the Control Human Calpain-1, warm all reagents and samples to room temperature before use. It is recommended that all standards, controls, and samples be assayed in duplicate. Biological samples should also be assayed with inhibition buffer.

          1. Remove the 96-Well Plate from the foil pouch.
          2. Add 100 µl Activation Buffer to designated wells. For biological samples, prepare additional wells by adding 100 µl Inhibition Buffer to designated wells.
          3. Add 50 µl diluted Standards, controls, and samples to designated wells.
          4. Add 50 µl Diluted Substrate to each well.
          5. Incubate for 15 min at room temperature and read the fluorescence using a fluorescence plate reader at an excitation wavelength of ~360-380 nm and an emission wavelength of ~440-460 nm.
          6. Calculate the results: Note: The data obtained can be displayed in two ways.
          a. As relative fluorescence units (RFU): Correct the fluorescence value of all samples by subtracting the value of the Blank and then calculate the mean fluorescence value for each sample from duplicate readings.
          b. As µmole AMC: Calculate the mean fluorescence value as stated above. Plot a graph correlating the mean fluorescence values of the Diluted Standards (y-axis) to the concentration of AMC (x-axis). The activity of calpain in samples can be displayed as the amount of free AMC released per time per mg protein.

          Note: For biological samples the fluorescence determined with Inhibition Buffer should be subtracted from the readings obtained in Activation Buffer, Final.
          Assay characteristics and examples

          Figure 1: Calpain-1 Activity

          Activity of native calpain 1 (Cat. No. 208713) was determined using 0.2 mM Suc-LLVY AMC in Assay Buffer, pH 7.4, in the presence of CaCl2 and the TCEP, according to the Detailed Protocol.


          Figure 2: Inhibitor Curve

          Inhibition of human calpain 1 by Z-LLY-FMK. The remaining activity of the enzyme was assayed with 0.2 mM Suc-LLVY AMC substrate at pH 7.4, in the presence of CaCl2 and the reducing agent TCEP. The activity was determined as indicated in the Detailed Protocol.


          Table 1: Calpain Activity in Various Cell Lysates

          Calpain activity in human cell lysates as measured as indicated in the Detailed Protocol. CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) was used to prepare cell lysates.

          Standard curve

          Figure 3: Typical Standard Curve

          Typical standard curve generated by using dilutions of the AMC Standard as indicated in the Detailed Protocol.

          Plate configuration

          Table 2: Sample Template

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