Specifications and Use
Fluorescent Substrate: Dabcyl-PChaGC(Me)HAK(5FAM)-NH2
Catalog Number: PEPDAB008m001, 1mg, 125 USD
Catalog Number: PEPDAB008m005, 5mg, 500 USD
Use:
This mmp substrate can be used to assess activity of enzymes in the MMP family. This mmp substrate has been used in enzymatic and cell based assays to assess activity of MMPs since it is specific for this family of metalloproteinases. It is the same substrate as described in publication 4 below by Bickett et al, except that it has Dabcyl/Fam as the fluorophore pair and is therefore more sensitive. It was originally described as a MMP9 substrate and MMP1 substrate. It demonstrates reasonably strong activity against all of those enzymes, with specificity constants, kcat/Km (M-1s-1), ranging from approximately 103 to 106. See our Product Sheets for its substrate specificity profile. Typically, the peptide is dissolved in DMSO to make a stock solution of about 10mM concentration. When used for in vitro assays, the substrate is often used at about 10uM concentration. Remember to keep the DMSO concentration in the final reaction at 1% or below, to avoid DMSO effects on the reaction, and remember to have an equivalent percentage of DMSO in the background wells. For use with the MMPs, the buffer should contain 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM CaCl2, 5 µM ZnSO4, and 0.01% Brij-35. Excitation and emission wavelengths are 485 and 530 nm respectively.
Molecular Weight:
1388.3 g/mol
Purity:
Greater than 95% as assessed by HPLC and Mass Spectrometry.
Solubility:
1 mg/ml in water with 10% Formic acid
Appearance:
Red lyophilized powder
Shipping
The peptide powder is shipped at room temperature.
Storage:
Upon receiving, the peptide should be stored at -70 oC. Avoid repeated freeze-thaw cycles. If dissolved in liquid (such as DMSO), aliquot into separate tubes to minimize the number of freeze-thaw cycles.
Stability:
Samples are sensitive to light and stable up to 6 months at -70oC.
References:
1. Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening.Marcia L Moss, Fred H Rasmussen.(2007) Analytical Biochemistry;366(2):144-8. PMID 17548045
2.Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities. Miles A Miller, Layla Barkal, Karen Jeng, Andreas Herrlich, Marcia Moss, Linda G Griffith, Douglas A Lauffenburger Integrative Biology 12/2010; 3(4):422-38.
3 .Potential of Fluorescent Metalloproteinase Substrates for Cancer Detection.Roopali Roy,1,5 David Zurakowski,2,5 Susan Pories,3 Marcia L. Moss,4,* and Marsha A. Moses1,5Clin Biochem. 2011 December; 44(17-18): 1434–1439.
4. A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9). Bickett DM, Green MD, Berman J, Dezube, Howe AS, Brown PJ, Roth JT, McGeehan GM, Anal Biochem. 1993 Jul;212(1):58-64.