Calpain Activity Assay Kit, Fluorogenic

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.

Assay Time: 1.5 h

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.

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.

Positive Control
Calpain 1

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.

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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.

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

Toxicity: Multiple Toxicity Values, refer to MSDS (O)