Recommended Products
usage
sufficient for 96 tests
Quality Level
packaging
pkg of 1 96-well plate(s)
manufacturer/tradename
Calbiochem®
storage condition
OK to freeze
avoid repeated freeze/thaw cycles
protect from light
input
sample type serum
sample type plasma
sample type cell lysate
detection method
fluorometric
shipped in
wet ice
General description
Calpains 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.
Components
Calibration 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.
Warning
Toxicity: Multiple Toxicity Values, refer to MSDS (O)
Specifications
Assay Time: 1.5 h
Preparation Note
1. 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.
Note: 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 µMExample: 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.
Storage and Stability
Upon 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.
Analysis Note
Positive Control
Calpain 1
Calpain 1
Other Notes
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. Oncogene17, 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.
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. Oncogene17, 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.
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.
Legal Information
CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany
Signal Word
Danger
Hazard Statements
Precautionary Statements
Hazard Classifications
Repr. 1B
Storage Class Code
6.1C - Combustible acute toxic Cat.3 / toxic compounds or compounds which causing chronic effects
Certificates of Analysis (COA)
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Martin K Childers et al.
Frontiers in pharmacology, 2, 89-89 (2012-02-01)
Calpains likely play a role in the pathogenesis of Duchenne muscular dystrophy (DMD). Accordingly, calpain inhibition may provide therapeutic benefit to DMD patients. In the present study, we sought to measure benefit from administration of a novel calpain inhibitor, C101
Ji-Youn Youn et al.
Circulation research, 104(1), 50-59 (2008-11-29)
Calpain was recently reported to mediate vascular endothelial growth factor (VEGF)-induced angiogenesis. In the present study, we investigated detailed molecular mechanisms. VEGF (100 ng/mL) induced a marked increase in endothelial cell production of NO(*), specifically detected by electron spin resonance.
Narci C Teoh et al.
PloS one, 9(9), e104376-e104376 (2014-09-16)
Ischemia-reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and
Juliet A Emamaullee et al.
Diabetes, 57(6), 1556-1566 (2008-03-22)
Clinical islet transplantation can provide insulin independence in patients with type 1 diabetes, but chronic graft failure has been observed. This has been attributed in part to loss of >or=60% of the transplanted islets in the peritransplant period, resulting in
Mariam Al-Omari et al.
Molecular medicine (Cambridge, Mass.), 17(9-10), 865-874 (2011-04-16)
A rapid recruitment of neutrophils to sites of injury or infection is a hallmark of the inflammatory response and is required for effective host defense against pathogenic stimuli. However, neutrophil-mediated inflammation can also lead to chronic tissue destruction; therefore, a
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