fluorogenic cathepsin v substrate  (BPS Bioscience)

Journal: BMC Biotechnology

Article Title: Novel concept microarray enabling PCR and multistep reactions through pipette-free aperture-to-aperture parallel transfer

doi: 10.1186/1472-6750-10-71

Figure Lengend Snippet: Selection of cathepsin E (CE)-inhibitory peptides . (a) An MMV plate plan adopted for the selection of CE-inhibitory peptides. First, magnetic beads were added to MMV1 and transferred to MMV2. MMV2 was changed with a cell-free transcription/translation solution. Independently, MMV3 was subjected to amplification of DNA (PCR reaction) and the products were transferred to MMV2. After transcription and translation reactions in MMV2, generated proteins were treated with restriction protease Xa, releasing peptides from the protein bound on the magnetic bead. After removal of magnetic beads, the supernatant was transferred to MMV5 which contains cathepsin E and its fluorogenic substrate. After incubation, the fluorescent product was monitored with a fluoroimager. The symbol 'arrow with a spiral' means that the preceding MMV is layered upside down as a donor MMV to the following one (recipient MMV). (b) Selection of CE-inhibitory peptides. In this case, dark wells contain CE-inhibitory peptides which block the CE enzyme reaction (generation of fluorescent products) and result in no production of fluorescent products. The arrows in inset indicate the inhibitory peptide-containing wells.

Article Snippet: A newly made MMV was treated to equilibrate with a permeation solution (50 mM sodium acetate, 0.1 M NaCl, pH 4.5) containing 5 μM fluorogenic substrate of cathepsin E (CE) MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Leu-Lys(DnP)-D-Arg-NH 2 (Code 3200-V, Peptide Institute, Inc., Osaka), and was half-filled with a CE reaction solution (permeation solution containing 5 μM substrate and 5 pmol CE additively).

Techniques: Selection, Magnetic Beads, Amplification, Generated, Incubation, Blocking Assay