Analysis Note

Positive ControlCalpain 1

Components

96-Well Plate, Calpain 1 Control, Substrate, Assay Buffer, Activation Buffer, Inhibitor, Reducing Agent, Plate Sealer and a user protocol.

General description

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

Legal Information

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

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.

Packaging

1 kit in Glass bottle

Preparation Note

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 Buffer2. Add 50 µl of the 1:50 dilution (step 1) to 4.95 ml Activation Buffer with Reducing Agent.

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

Principle

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 Stability

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:Refrigerate (4°C): Assay Buffer, Activation Buffer, Substrate, Inhibitor, and 96-well plate r>Freezer (-20°C): Reducing Agentr>Freezer (-70°C): Control, Human Calpain 1r>r>Note: Following initial use the Control, Human Calpain 1 should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles.

Warning

Toxicity: Irritant (B)