Elastase assays.
Two methods of P. aeruginosa elastase purification are described: Method 1 involves concentration of sample supernatants, followed by DEAE-Sepharose liquid chromatography, whereas Method 2 involves initial fractionations followed by molecular sieving and hydrophobic interaction high-performance liquid chromatography. The choice of methods depends on the available equipment and supplies. The methods of assaying elastase activity described as useful for a variety of applications. The elastin-nutrient agar plate method is a qualitative assay to determine the presence of elastase activity produced by a given culture or colony. Use of the quantitative elastin-Congo red assay is appropriate for determining elastase activities of mid-to-high elastase-producing cultures. For more sensitive determinations of P. aeruginosa elastase activity, use of the fluorogenic substrate is advisable.
Rust
L
L
Department of Microbiology and Immunology, University of Rochester, School of Medicine and Dentistry, New York 14642.
Messing
C R
CR
Iglewski
B H
BH
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification; D002417:Cattle; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D003224:Congo Red; D004549:Elastin / Q000378:metabolism; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010196:Pancreatic Elastase / Q000032:analysis* / Q000302:isolation & purification; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D009842:Oligopeptides; C044862:2-aminobenzoylalanyl-glycyl-leucyl-alanyl-4-nitrobenzylamide; D003224:Congo Red; D004549:Elastin; D010196:Pancreatic Elastase