By John K. Spitznagel (auth.), William M. Shafer (eds.)
In Antibacterial Peptide Protocols, major specialists evaluate for the 1st time in a single quantity all of the significant biochemical, molecular, bacteriological, and actual ideas on hand to evaluate antimicrobial peptides. those cutting-edge tools make certain simply reproducible ends up in such vital techniques because the isolation and characterization of antimicrobial peptides, the molecular characterization of genes encoding antimicrobial peptides, and using expression structures to isolate peptides. Bioassays and microbial genetic innovations also are incorporated, as are antibacterial assays because the ultimate readout system.
those equipment certain in Antibacterial Peptide Protocols will play an enormous function within the therapy of infectious illnesses, rather with the expanding challenge of multidrug-resistant microbes and the relative dearth of recent antibiotics being supplied through pharmaceutical companies.
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18. , Hetru, C , and Hoffmann, J. A (1992) A novel insect defensin mediates the inducible antibacterial activity in larvae of the dragonfly Aeschna cyanea (Paleoptera, Odonata). Eur J. Biochem 209, 977-984. , and Hoffmann, J. A (1993) Purification and characterization of a scorpion defensin, a 4 kDa antibacterial peptide presenting structural similarities with insect defensins and scorpion toxins. Biochem Biophys. Res. , Holder, F , Hetru, C , Hoffmann, J A , and Bulet, P. (1994) Novel inducible antibacterial peptides from a hemipteran insect, Pyrrhocoris apterus Biochem J.
Adjust the spore density to 2 x 10^ spores per mL. Transfer 4 (or more) x 600 ^L to sterilized microtubes. To each tube add 600 [iL of 50% glycerol Vortex each tube and transfer their contents in 100-pL aliquots in sterilized microtubes (see Note 14). 2 Antibacterial and Antifungal Assays Antimicrobial (antibacterial and antifungal) activity is monitored during the different purification steps by liquid growth inhibition assays. 1. ANTIFUNGAL ASSAY 1. Suspend spores in a growth medium containing potato dextrose broth (Difco, E.
11. 12. 13 14 15 16 17 45 214 and 225 nm (detection of the peptide bond, high sensitivity but absorption of solvent at this low wavelength is strong). It is preferable to collect the peaks by hand rather than using an automatic collector to minimize the number of purification steps. This collecting is highly facilitated by the use of an analogic direct recorder Each fraction is concentrated with vacuum centrifuge and an aliquot is then tested for antimicrobial activity. The size-exclusion column should be equilibrated in 30% acetonitrile in acidified water (TFA 0 05%) during several hours for a good stabilization of the phase.