Protein A, gold

Figure 4. Transmission electron micrograph showing tuber tissue labelled with protein A-gold. Localization of PL3 enzyme is indicated by arrow-heads. S= Starch grain CW= Cell wall Bar= SOOnm. x 20 000.

To remove excess protein A, centrifuge the preparation at a minimum of 50,000g for 30 minutes to several hours (4°C), depending on the size of the particles and the amount of solution. Discard the supernatant, and resuspend the protein A-gold pellet in 0.01M sodium phosphate, pH 7.4, containing 1 percent PEG. 

Bendayan, M. (1989) Ultrastructural localization of insulin and C-peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high-resolution protein A-gold approach. Am. J. Anat. 185, 205-216. 

Bendayan, M., and Garzon, S. (1988) Protein G-gold complex Comparative evaluation with protein A-gold for high-resolution immunocytochemistry. J. Histochem. Cytochem. 36, 597-607. 

Hearn, S.A. (1987) Electron microscopic localization of chromogranin A in osmium-fixed neuroendocrine cells with a protein A-gold technique./. Histochem. Cytochem. 35, 795-801. 

Lethias, C., Hartmann, D.J., Masmejean, M., Ravazzola, M., Sabbagh, I., Ville, G., Herbage, D., and Eloy, R. (1987) Ultrastructural immunolocalizadon of elastic fibers in rat blood vessels using the protein A-gold technique./. Histochem. Cytochem. 35, 15-21. 

Yokota, S. (1988) Effect of particle size on labeling density for catalase in protein A-Gold immunocyto-chemistry./. Histochem. Cytochem. >6, 107-109. 

Bendayan M, Zollinger M. Ultrastractural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J Histochem Cytochem 1983 31 101-109. 

Roth J, Bendayan M, Orci L (1978) Ultrastructural localization of intracellular antigens by the use of protein A gold complex. J Histochem Cytochem 26 1074 1081... 

Bendayan, M. (1984) Protein A-gold electron microscopic immunocytochemistry methods, applications, and limitations. J. Electron Micros. Tech. 1, 243-270. 

Roth, J. (1982) The preparation of protein A-gold complexes with 3 nm and 15 nm gold particles and their use in labeling multiple antigens on ultra-thin sections. Histochem. J. 14,791-801. 

Bendayan, M. (1989) Protein A-gold and protein G-gold postembedding immuno-electron microscopy, in Colloidal Gold, vol. 1 (Hayat, M. A., ed.). Academic, New York, pp. 34-96. 

Roth, J. (1982) The protein A-gold (pAg) technique—a qualitative and quantitative approach for antigen localization on thin sections, in Techniques in Immuno-cytochemistry, vol. 1 (Bullock, G. R. and Petrusz, P., eds.), Academic, New York, pp. 107-154. 

Roth, J. (1984) The protein A-gold technique for antigen localization in tissue sections by light and electron microscopy, in Immunolabelling for Electron Microscopy (Polak, J. M. and Vamdell, I. M., eds.), Elsevier, New York, pp. 113-122. 

Wipe excess liquid from the area around the sections, and apply sufficient 5% heat-inactivated normal serum from the second antibody species to cover each entire section (15 min). If protein A-gold probes are used, flood the sections with BSA-PBS rather than normal serum. It is important to keep the sections covered and in a humid environment throughout all incubations, preferably using a purpose-made humidity chamber. Depending on the size of the section, about 50-200 uL of reagent is sufficient to cover each preparation... 

Mannweiler, K., Hohenberg, H., Bohn, W., and Rutter, G. (1982) Protein A-gold particles as markers in replica immunocytochemistry. high resolution electron microscope investigations of plasma membrane surfaces J Microsc 126,145-149... 

Herbener, G. H. (1989) Use of the protein A—gold immunocytochemical and enzyme—gold cytochemical techniques in studies of vitellogenesis. Am. J. Anat. 185,244—254. Hermanson, G. T., Mallia, A. K., and Smith, P. K. (1992) in Immobilized Affinity Ligand Techniques. Academic Press, San Diego. 

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