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48-602MAG
Buffer Detection Kit for Magnetic Beads
1 Kit
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Description
Overview
A highly sensitive and specific assay for measuring Calpain 1 and 2 activity in biological samples like cell lysate or tissue extracts and for screening enzyme inhibitors. Calpains (calcium ion-dependent papain-like cysteine proteases) are widely distributed in all mammalian cells. They mediate calcium-induced activation of the Erk1/Erk2 MAPK pathway and are believed to participate in intracellular signal processing via limited proteolysis of their targets.
• Sensitive: detects calpain activity at 63-1000 ng/ml
• Specific: Substrate and inhibitor optimized for specific detection of calpain activity
• Versatile: Homogeneous 96-well format can be used with biological samples or for inhibitor screens
• Convenient: Contains all reagents to assay samples in 30 min
Catalogue Number
CBA054
Brand Family
Calbiochem®
Application Data
The activity of Calpain 1, Human Erythrocytes (Cat. No. 208713) was determined as outlined in the Detailed Protocol provided above. The activities of Calpain 2, Porcine Kidney (Cat. No. 208715) and Calpain 2, Rat, Recombinant, High Purity, E. coli (Cat. No. 208718) were determined as outlined in the Detailed Protocol provided above. Calpain 1, Human Erythrocytes (Cat. No. 208713) and Calpain 2, Porcine Kidney (Cat. No. 208715) were incubated with Z-LLY-FMK (Calpain Inhibitor IV, Cat. No. 208724) at room temperature for 10 min. Following incubation the activities were determined as outlined in the Detailed Protocol provided above.
Materials Required but Not Delivered
• CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) • Protein assay (Non-Interfering Protein Assay™, Cat. No. 488250 or BCA Protein Assay, Cat. No. 71285) • Pipettors or multi-channel pipettor carefully calibrated to the target volume • Fluorescence plate reader capable of measuring fluorescence at an excitation wavelength of 320 nm and an emission wavelength of 480 ± 20 nm
References
References
Higuchi, M., et al. 2005. J. Biol. Chem.280, 15229; Tompa, P., et al. 2004. J. Biol. Chem.279, 20775; Veeranna, et al. 2004. Am. J. Pathol.165, 795; Dainese, E., et al. 2002. J. Biol. Chem.277, 40296; Wang, K.K., et al. 2000. Trends Neurosci.23, 20; Black, R.A., et al. 1997. Nature385, 729; Molinari, M. and Carafoli, E. 1997. J. Membr. Biol.156, 1; Sorimachi, H., et al. 1997. Biochem. J.328, 721.
Product Information
Detection method
Fluorometric
Form
96 Tests
Format
96-well plate
Kit contains
96-Well Plate, Calpain 1 Control, Substrate, Assay Buffer, Activation Buffer, Inhibitor, Reducing Agent, Plate Sealer and a user protocol.
63-1000 ng/ml as measured with purified human calpain 1.
Sample Type
Cell lysates, tissues extracts
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Intended use
The Calbiochem® InnoZyme™ Calpain 1 & 2 Activity Kit is designed to measure Calpain 1 and 2 activity in biological samples such as cell lysates or tissue extracts and to screen enzyme inhibitors.
Storage and Shipping Information
Ship Code
Dry Ice Only
Toxicity
Irritant
Storage
Multiple storage requirements
Storage Conditions
Upon arrival store the Control, Human Calpain 1 at -70°C and the remaining components at -20°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
Higuchi, M., et al. 2005. J. Biol. Chem.280, 15229; Tompa, P., et al. 2004. J. Biol. Chem.279, 20775; Veeranna, et al. 2004. Am. J. Pathol.165, 795; Dainese, E., et al. 2002. J. Biol. Chem.277, 40296; Wang, K.K., et al. 2000. Trends Neurosci.23, 20; Black, R.A., et al. 1997. Nature385, 729; Molinari, M. and Carafoli, E. 1997. J. Membr. Biol.156, 1; Sorimachi, H., et al. 1997. Biochem. J.328, 721.
Upon arrival store the Control, Human Calpain 1 at -70°C and the remaining components at -20°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
Note: Following initial use the Control, Human Calpain 1 should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles.
Intended use
The Calbiochem® InnoZyme™ Calpain 1 & 2 Activity Kit is designed to measure Calpain 1 and 2 activity in biological samples such as cell lysates or tissue extracts and to screen enzyme inhibitors.
Background
Calpains, calcium ion-dependent papain-like cysteine proteases, are widely distributed in all mammalian cells.
Calpain 1 and 2 are heterodimeric proteins comprised of a 78-80 kDa catalytic subunits encoded by the capn 1 and 2 genes, and a 28-30 kDa regulatory subunit, common to both enzymes, encoded by the capn4 gene. In vitro, calpain 1 (µ-calpain) and 2 (m-calpain) are activated by micromolar (10-50 µM) or millimolar (0.2-0.35 mM) Ca2+, respectively. There is evidence to suggest that calpains are tightly regulated post-transcriptionally by calpastatin (the endogenous calpain inhibitor), calcium requirements, and auto-proteolytic cleavage.
Calpains are believed to participate in intracellular signal processing via limited proteolysis of their targets. As irreversible modifiers of a variety of proteins, calpains are likely to be involved in the regulation of many life processes. They may play a central role in the execution of apoptosis either upstream or downstream of caspases in glucocorticoid-treated and irradiated thymocytes, neuronal cells exposed to various stresses, and MCF-7 breast cancer cells treated with β-lapachone. Caspases and calpains share several substrates (e.g., calpastatin, fodrins, focal adhesive kinase, calmodulin-dependent kinases, actin, vimentin, and keratins), suggesting that these enzymes may execute cell death in a partially indistinguishable manner. Calpains also mediate calcium-induced activation of the ERK1/2 MAPK pathway resulting in aberrant phosphorylation of the neuronal cytoskeleton, an early event in Alzheimer's disease.
Principles of the assay
The Calbiochem® InnoZyme™ Calpain 1 & 2 Activity Kit is a sensitive assay optimized for measuring active calpain 1 and 2. The Substrate used in the assay (DABCYL)-TPLK;-;SPPPSPR-(EDANS) is based on the finding that the amino acid preferences for calpain recognition/cleavage extend to 11 positions around the scissile amide bond. Calpain-containing samples are incubated with the Substrate in the presence of calcium ions and Reducing Agent. Cleavage of the scissile amide bond, K-S, releases the fluorophore (EDANS) from the internal quenching molecule (DABCYL), resulting in an increase in fluorescence. Fluorescence is measured at excitation wavelength of 320 nm and an emission wavelength of 480 ± 20 nm. All biological samples are assayed in parallel with the calpain inhibitor, Z-LLY-FMK (included).
Materials provided
• 96-well Plate (Kit Component No. JA7666-1EA): 96-well plate, supplied as six 2 x 8 well strips • Control, Human Calpain 1 (Kit Component No. JA9360-20UL): 1 vial, 20 µl supplied at 1 µg/µl • Substrate (Kit Component No. JA9183-200UL): 1 vial, 200 µl, supplied as 2 mM (DABCYL)-TPLKSPPPSPR-(EDANS) in DMSO • Assay Buffer (Kit Component JA7667-25ML): 1 bottle, 25 ml • Activation Buffer (Kit Component No. JA7663-20ML): 1 bottle, 20 ml • Inhibitor (Kit Component No. JA9184-20UL): 1 vial, 20 µl, supplied as 1 mM Z-LLY-FMK in DMSO • Reducing Agent (Kit Component No. JA7665-100UL): 1 vial, 100 µl, TCEP • Plate Sealer (Kit Component No. JB155-EA): 1 each
Materials Required but not provided
• CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) • Protein assay (Non-Interfering Protein Assay™, Cat. No. 488250 or BCA Protein Assay, Cat. No. 71285) • Pipettors or multi-channel pipettor carefully calibrated to the target volume • Fluorescence plate reader capable of measuring fluorescence at an excitation wavelength of 320 nm and an emission wavelength of 480 ± 20 nm
Preparation
Dilute samples with Assay Buffer as needed.
Note: Calpain is a temperature sensitive enzyme. Dilutions of calpain control must be prepared using chilled Assay Buffer and kept on ice at all times.
Guidelines for sample preparation:
• The recommended protein concentration for cell lysates is ≥2 mg/ml as determined using a standard protein assay (e.g. Non-Interfering Protein Assay™, Cat. No. 488250 or BCA Protein Assay, Cat. No. 71285)
• The recommended dilution factor for cell lysates with a protein concentration of ≥2 mg/ml is ~5 or higher
Reagent preparation
All reagents necessary to perform the assay are supplied with the kit. Warm all reagents (except Calpain Control and Assay Buffer) to room temperature (15-25°C) just prior to use. The following reagent preparation guidelines will result in sufficient volume for one 96-well plate.• Control, Human Calpain 1, diluted: Dilute the Control, Human Calpain 1 100-400-fold with chilled Assay Buffer; maintain on ice at all times.
• Activation Buffer with Reducing Agent: Add 80 µl Reducing Agent to 9.92 ml Activation Buffer.
• Substrate (1X): Dilute Substrate 1:25 by adding 200 µl Substrate to 4.8 ml Assay Buffer
• Inhibitor (1X): Dilute the Inhibitor as follows:
1. Dilute the Inhibitor 1:50 by adding 2 µl Inhibitor to 98 µl Assay Buffer
2. Add 50 µl of the 1:50 dilution (step 1) to 4.95 ml Activation Buffer with Reducing Agent.
Detailed protocol
It is recommended that all samples and controls be assayed in duplicate.
1. Remove the desired number of strips from the 96-Well Plate, return the remaining strips to foil pouch, and close the re-sealable edge. 2. Add 50 µl Control, Human Calpain 1, and diluted samples to designated wells. Prepare a blank by adding 50 µl Assay Buffer to a designated well. 3. Add 100 µl Activation Buffer with Reducing Agent. Cover the plate with the Plate Sealer and incubate 2-3 min at room temperature with gentle shaking. Note: All biological samples should be assayed in parallel with the Activation Buffer with Reducing Agent and Inhibitor. 4. Add 50 µl Substrate (1X) to each well and incubate 30 min at room temperature in the dark with gentle shaking. 5. Read the fluorescence at an excitation wavelength of 320 nm and an emission wavelength of 480 ± 20 nm. 6. The results are displayed in a 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 set of duplicate samples.
Assay characteristics and examples
Figure 1: Human calpain 1 activity
The activity of Calpain 1, Human Erythrocytes (Cat. No. 208713) was determined as outlined in the Detailed Protocol provided above.
Figure 2: Human Calpain 2 Activity
The activities of Calpain 2, Porcine Kidney (Cat. No. 208715) and Calpain 2, Rat, Recombinant, High Purity, E. coli (Cat. No. 208718) were determined as outlined in the Detailed Protocol provided above.
Figure 3: Inhibition of Calpain 1 and 2 by Z-LLY-FMK
Calpain 1, Human Erythrocytes (Cat. No. 208713) and Calpain 2, Porcine Kidney (Cat. No. 208715) were incubated with Z-LLY-FMK (Calpain Inhibitor IV, Cat. No. 208724) at room temperature for 10 min. Following incubation the activities were determined as outlined in the Detailed Protocol provided above.
Figure 4: Calpain 1 and 2 Activity in Biological Samples
Jurkat (human lymphoblastic acute T cell leukemia), HL-60 (human myoblastic acute promyelocytic leukemia), and SK-BR-3 (human breast adenocarcinoma) cells were cultured in DMEM or RPMI medium supplemented with 10% FCS and harvested at 80-90% confluency. To prepare total cell lysates, cells were pelleted, washed, and resuspended in Lysis Buffer (CytoBuster™ Protein Extraction Reagent, Cat. No. 71009) at 200-600 µl/107 cells, based on cell size. The suspension was incubated for 30-40 min on ice and centrifuged for 10 min at 15,000 x g, 4°C. The supernatant was used immediately or dispensed into aliquots and frozen at -70°C for future assay. Human spleen extract was prepared by homogenizing tissue with 170 mM NaCl, 2 mM EDTA. The protein concentration in cell lysates and tissue extracts was determined using the BCA protein assay. Calpain activity was determined as outlined in the Detailed Protocol provided above.
Assay Range
63-1000 ng/ml as measured with purified human calpain 1.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. Interactive Pathways™, InnoZyme™ and CytoBuster™ are trademarks of EMD Chemicals, Inc. Non-Interfering Protein Assay™ is a trademark of Geno Technology, Inc.
