tailieunhanh - Atomic Force Microscopy Episode 2 Part 5

Tham khảo tài liệu 'atomic force microscopy episode 2 part 5', kỹ thuật - công nghệ, cơ khí - chế tạo máy phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | 286 Smith and Benos Fig. 2. Representative images illustrating the localization of epithelial sodium channels by AFM. A Image of the surface of an A6 renal epithelial cell labeled with colloidal gold particles conjugated to nonimmune IgG showing the microvilli. B Image of the surface of an A6 cell labeled with colloidal gold particles conjugated to an anti-epithelial sodium channel antibody showing localization of marker to the microvilli. Note marked increase in height of microvilli when compared to A. Reproduced with permission from the American Physiological Society from ref. 2. AFM Localization of ENaC 287 2. Operate Bioscope in the contact mode. After engagement with the sample adjust the scan force to values in the range of 5 nN. Record images with a display of 512 lines screen 1 pm2 scan area at a constant force. Typical area rate of 1-2 Hz. 3. Scan multiple cells cover slip and multiple cover slips labeled with both antibody and control IgG. Also scan cover slips bearing cells that were fixed but not labeled to obtain dimensions and surface topography of the cells before antibody treatment. Binding of colloidal gold antibody conjugates to the epitope-tagged ENaC results in a marked difference in height of the cell surface when compared to controls see Fig. 2 Note 5 . 4. Notes 1. cDNAs have been cloned for the a p and Y subunits of Xenopus 9 rat 10-13 mouse 14 and human ENaC 15-17 . All species can be expressed heterologously in mammalian cells. Typically the cDNAs are available upon request from the laboratories that cloned them. Subcloning of the subunits into a mammalian expression vector such as may be required. A number of laboratories have produced constructs for ENaC subunits with extracellular epitope tags that may be available upon request 3 4 18 . 2. Although we produce colloidal gold particles following the method of Slot and Geuze 7 colloidal gold particles produced by this method are available commercially Sigma Electron Microscopy .

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