Analysis Note

Positive ControlMMP-9 (Cat. No. PF140)

Components

Gelatinase Substrate, pro-MMP-2 Positive Control, Inhibitor, APMA, Assay Buffer, 96-Well Plate, Plate Sealer, and a user protocol

General description

A sensitive fluorogenic assay (Exc. max: ~325 nm; Emi. max.: ~393 nm) for the measurement of gelatinases (MMP-2 and MMP-9). Also useful for the screening of gelatinase inhibitors.

Legal Information

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

Other Notes

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.

Lauer-Fields, J.L., et al. 2003. J. Biol. Chem.278, 18140.Chang, C., and Werb, Z. 2001. Trends Cell Biol.11, S37.Kleiner, D.E., and Stetler-Stevenson, W.G. 1999. Cancer Chemother. Pharmacol.43 (suppl.), S42.Cockett, M.I., et al. 1998. Biochem. Soc. Symp.63, 295.Olson, M.W., et al. 1997. J. Biol. Chem.272, 29975.Will, H., et al. 1996. J. Biol. Chem.271, 17119.MacDougall, J.R., et al. 1995. Cancer Metastasis Rev.14, 351.Liotta, L.A., et al. 1990. Semin. Cancer Biol.1, 99.

Preparation Note

All samples should be diluted in Activation Buffer just prior to performing the assay. The recommended dilution for serum/plasma is 1:5-1:10; the recommended dilution for culture supernatant is 1:3-1:5.

Warm all reagents, except the positive control, to room temperature prior to performing the assay. The Gelatinase (MMP-2/MMP-9) Control should be kept on ice until use. Activation Buffer: Add 5 µl p-Aminophenylmercuric Acetate (APMA) to a tube containing 5 ml Assay Buffer. If desired, a smaller or larger volume of Activation Buffer can be prepared; the ratio of APMA to Assay Buffer should be 1:1000. Note: the p-Aminophenylmercuric Acetate (APMA) can be stored at –20°C and thawed again for future use. The Activation Buffer should be made fresh for each application and discarded after use. Substrate Working Solution: Dilute the Gelatinase (MMP-2/MMP-9) Substrate 1:5 with dH₂O. For example, to prepare enough reagent for one 16-well strip mix 40 µl Gelatinase (MMP-2/MMP-9) Substrate with 160 µl dH₂O. Note: the Gelatinase (MMP-2/MMP-9) Substrate can withstand several freeze/thaw cycles, but it is recommended that it be dispensed into small amounts (~20 µl) for maximum stability. Inhibitor Working Solution: Dilute the Gelatinase (MMP-2/MMP-9) Inhibitor 1:50 with Activation Buffer. For example, to prepare enough for 5 wells, mix 5 µl Gelatinase (MMP-2/MMP-9) Inhibitor with 245 µl Activation Buffer. Gelatinase (MMP-2/MMP-9) Control: The Gelatinase (MMP-2/MMP-9) Control must be activated prior to performing the assay. Add 1 µg of Gelatinase (MMP-2/MMP-9) Control to 400 µl Activation Buffer to yield a 2.5 µg/ml solution. Prepare serial dilutions of the reconstituted/activated Gelatinase (MMP-2/MMP-9) Control in Activation Buffer ranging from ~20 to 2000 ng/ml; these dilutions can be used to generate a standard curve for the assay. Note: the reconstituted Gelatinase (MMP-2/MMP-9) Control can be dispensed into smaller aliquots (5 or 10 µl) and stored for several months at -20°C.

Principle

The Calbiochem™ InnoZyme™ Gelatinase (MMP-2/MMP-9) Activity Assay Kit is designed for measuring gelatinase (MMP-2 and MMP-9) activity in a variety of samples. It is most suitable for screening gelatinase inhibitors using purified enzymes. It is not as sensitive as zymography, but it is still sufficient for measuring activity in biological samples (e.g., serum and plasma), culture supernatant, or tissue extracts. The assay utilizes a triple-helical peptide substrate that is highly selective for MMP-2 and MMP-9.

Specifications

Assay Time: 2 - 6 h

Storage and Stability

Upon arrival store the MMP-2 at -70°C and all other contents of the kit at -20°C.

Warning

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