By Pontus Nordenfelt, Mattias Collin
This quantity discusses a number of equipment and protocols used for the experimentation of a variety of bacterial species, comparable to Streptococcus pyogenes, Staphylococcus aureus, Streptococcus pneumonia, Listeria monocytogenes, and Mycobacterium marinum. Bacterial Pathogens: tools and Protocols is split into 6 components: half 1 describes diverse techniques to choosing and characterizing bacterial effector molecules; half 2 offers with structural biology of bacterial pathogenesis and the way to beat folding and balance issues of recombinantly expressed proteins; half three information technique that identifies micro organism in complicated groups and the way genomes of bacterial pathogens have advanced; half four displays at the speedy improvement of complicated imaging suggestions that deal with questions on molecular houses of person reside micro organism, ultrastructure of surfaces, and subcellular localization of bacterial proteins; half five describes equipment from in vitro and in vivo modeling of bacterial infections; and half 6 explores how bacterial pathogens are the genuine specialists of the immune procedure. Written within the hugely winning Methods in Molecular Biology sequence layout, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and tips about troubleshooting and warding off identified pitfalls.
Cutting-edge and finished, Bacterial Pathogens: tools and Protocols is a helpful source for a person who's drawn to this interesting and evolving field.
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Extra info for Bacterial Pathogenesis: Methods and Protocols
Radiolabeled ligand in binding buffer: calculate the volume of radiolabeled ligand that will result in 150 μCi activity/50 mL of buffer (see Note 3 regarding radiolabel mixture). To this volume add 5 mL 10× binding buffer and ddH2O to 50 mL in a 50 mL conical. Store at −20 °C until use. 2. 01 % (vol) Tween 20 in H2O: mix 5 μL Tween 20 and 50 mL ddH2O in a 50 mL conical. Make fresh each time. 30 Mona W. Orr and Vincent T. Lee 3. 96-well pin tool with 2 μL slot (V&P Scientific). 4. 45 μm dry nitrocellulose membrane sheets cut to a size that permits duplicate stamps on one sheet (12 cm × 19 cm).
C400485200 9. Kronvall G, Simmons A, Myhre EB, Jonsson S (1979) Specific absorption of human serum albumin, immunoglobulin A, and immunoglobulin G with selected strains of group A and G streptococci. Infect Immun 25:1–10 10. Kahn F, Mörgelin M, Shannon O et al (2008) Antibodies against a surface protein of Streptococcus pyogenes promote a pathological inflammatory response. PLoS Pathog 4:e1000149. 1371/journal. 1000149 11. Nordenfelt P, Waldemarson S, Linder A et al (2012) Antibody orientation at bacterial surfaces is related to invasive infection.
This section uses radiation and should be performed in an area certified for use of radioactivity. 1. Lay out a pre-cut nitrocellulose sheet on the radiation bench so that the 12 cm edge is parallel to the front edge of the bench (see Note 9). 2. 4, item 1) (see Note 3). This can be thawed in a beaker of warm water to speed the process. If all wells show positive binding, there may be nonspecific binding proteins in the lysates (see Note 10). 3. 01 % Tween 20 into a reservoir large enough to accommodate the pin tool.