Testing of the P aeruginosa Isolate for Exotoxin A Production by Western Blotting
Special growth media was prepared to maximize exotoxin A yields. Trypticase soy broth (Baltimore Biological Laboratory, Cokey sville, MD), 38 g/L, was iron depleted by adding the chelating agent, Chelex 100 (Bio-Rad, Rockville, Centre, NY), 10 g/L, followed by stirring for 12 h at 5°C. The broth was then filtered through Whatman No. 2 filter paper before undergoing ultrafiltration using a 10,000-MW cutoff membrane (AMICON, Danver, MA). The media was supplemented with glycerol 1 percent and monosodium glutamate 0.05 mol/L. Nitrilotriacetic acid (Sigma Chemical Co., St. Louis, MO) was added (1.9 g/L), the pH was adjusted to 7.0, and the broth was sterilized. buy ortho tri-cyclen
Our experimental strain of P aeruginosa and strain PA-103 were inoculated into separate aliquots of the special media. PA-103 is a known exotoxin A producer and was used as a positive control to validate our methods. After overnight growth, both cultures were centrifuged and the supernatants were decanted. The supernatants were diluted 1:10 (V:V) with “stacking gel” buffer containing 125 mmol/L of Tris-HCL, at a pH of 6.8, and then concentrated 50-fold by ultrafiltration using 10,000-MW cutoff membranes (Amicon, Danvers, MA).
Category: Pseudomonas aeruginosa
Tags: aeruginosa, endotoxin, sepsis, septic shock