ProteaseSpots™

Benefits of the ProteaseSpots™ System

• Screen large numbers of substrates economically
• You specify the individual substrate sequences
• Obtain semi-kinetic data
• Easy handling in 96-well plates

Applications for ProteaseSpots™

• Detection of protein cleavage sites utilizing peptide scans
• Identification of novel substrates with de novo libraries
• Measurement of protease activities with known substrates
• Characterization of cleavage sites through substitutional analyses

Figure:
The protease assay is based on peptides bound to small cellulose discs in 96-well microtiter plates. These peptides bear a fluorescent moiety at the N-terminus. After incubation with the active protease, aliquots are transferred to an additional plate for detection of the N-terminally labeled peptide fragment
(excitation: 325 nm, emission: 420 nm).

Figure: Fluorescence intensities of a caspase-3 substrate VDQMDGW single site L-amino acid substitutional analysis. This method identifies new or improved substrates. Each residue in the sequence (rows) was replaced by all 20 amino acids (columns).

Figure:
Fluorescence intensity of trypsin cleaved peptides derived from hen egg lysozyme. The entire sequence was synthesized using 7-mer overlapping peptides shifted by three amino acids.

Related products and services

Protease Substrate Sets
Internally quenched (Dabcyl/EDANS) protease substrate collections ready-to-screen in microtiter plates.

Protease Peptide Microarrays
Compilation of protease substrate peptides on ready-to-use PepStar™ peptide microarrays.

Protease Profiling Service
Profiling service for protease using high-content PepStar™ peptide microarrays.

References:

• Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8, Naus et al., Biol Chem.
  2006, Vol. 387, 3 (2006), p. 337-46

• Screening a combinatorial peptide library to develop a human glandular kallikrein-2 activated prodrug as targeted therapy for prostate cancer, Janssen et al, Mol. Cancer Ther., Vol. 3 (2004), p. 1439–1450 (abstract)

• Processing of the Human Transferrin Receptor at Distinct Positions within the Stalk Region by Neutrophil Elastase and Cathepsin G, Kaup et al., Biol. Chem., Vol. 383 (2002), p. 1011-1020 (abstract)

• Escherichia coli DegP protease cleaves between paired hydrophobic residues in a natural substrate: the PapA pilin, Jones et al, J. Bacteriol. (2002) p. 5672-5771(abstract)