By Frank DeLeo, Michael Otto
Bacterial infections have an effect on international future health at the present time as a number one explanation for morbidity and mortality. This publication provides in-depth tools and cutting-edge protocols for investigating particular mechanisms of pathogenesis for quite a lot of micro organism. Written via specialists within the box, this worthy assortment comprises protocols to check host-pathogen interactions, animal versions of an infection, and novel methods to selecting healing objectives designed to manage infections.
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Extra info for Bacterial Pathogenesis (Methods in Molecular Biology, v431)
2. Wash cell pellets with 1 mL of ice-cold TE buffer, resuspend in 1 mL TE buffer, and transfer into screw top tubes containing 500 μL of glass beads. 3. 5 m/s. 4. Centrifuge lysate at 21,000 × g for 25 min at 4 °C to remove cell debris and then transfer supernatant to a new tube. 5. Centrifuge at 21,000 × g for 45 min (all at 4 °C) to remove insoluble and aggregated proteins, which disturb the isoelectric focusing (IEF) of the proteins. 6. Determine protein concentration by using Roti-Nanoquant (Roth) (see Note 3).
20 Chaussee et al. 3. Centrifuge samples at 25,000 × g for 10 min at 4 °C. Transfer the supernatant fluid to a clean microcentrifuge tube. 4. Slowly pipet 125 μL (see Note 19) of the sample to the center of a 7-cm ceramic strip holder (see Note 20). Remove any bubbles. 5. Remove the protective plastic cover from the IPG strip. Position the IPG strip with the gel side down and the anodic end of the strip directed toward the anodic end of the strip holder. Lower the strip into the sample starting with the anodic end (see Note 21).
Each of the three protein extracts is labeled with one CyDye. After labeling, the three samples are mixed together and run on the same gel. In this way, the same protein labeled with the different CyDye will migrate to the same position on the 2D gel, which helps to limit errors due to experimental variation (see Fig. 4 and Color Plate 2, following p. 46). Each of the individual samples can be visualized independently by selecting the appropriate excitation and emission wavelength to scan for each CyDye (see Fig.