Specifications and Use
Fluorescent Substrate: Dabcyl-SPLAQAVRSSK(5FAM)-NH2
Catalog Number: PEPDAB010m001, 1mg, 125 USD
Catalog Number: PEPDAB010m005, 5mg, 500 USD
Use:
This fluorescent ADAM substrate was originally described by us in the publication, ” Fluorescent substrates useful as high-throughput screening tools for ADAM9″. This ADAM substrate is based on the cleavage sequence of precursor TNF-alpha and has been used to assess activity of ADAM17 in single cell assays as well as standard in vitro enzymatic and cell based assays (See publications below). See our Product Sheets for the substrate specificity profile of this substrate. This ADAM substrate is also an excellent substrate for ADAM9 and ADAM10. This substrate is not specific for ADAM family members as it can also be processed by members of the MMP family of proteinases. BioZyme Inc, does sell specific substrates for ADAM or MMP family members (Please see our Product Sheets or Catalog, for the substrate specificity profile). It demonstrates reasonably strong activity against all of those enzymes, with specificity constants, kcat/Km (M-1s-1), ranging from approximately 4 x 103 to 4 x 105. 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 well. For use with ADAM family members, the buffer should consist of 25mM Tris, pH 8, 10mM CaCl2 , and 6 x 10-4 Brij detergent. 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:
1751.9 g/mol
Purity:
Greater than 92% 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 stable up to 6 months at -70oC.
References:
1. Fluorescent substrates useful as high-throughput screening tools for ADAM9. Marcia L Moss, Fred H Rasmussen, Raphael Nudelman, Peter J Dempsey, Jason Williams Combinatorial chemistry & high throughput screening 12/2009; 13(4):358-65.
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.High-throughput protease activity cytometry reveals dose-dependent heterogeneity in PMA-mediated ADAM17 activation. Wu L, et al. Integr Biol (Camb). 2015 May;7(5):513-24. doi: 10.1039/c5ib00019j.