【正文】 reaction with the enzyme Figure 363. Immunogold electron microscopy. Electron micrographs of an insulinsecreting cell in which the insulin molecules have been labeled with antiinsulin antibodies bound to tiny colloidal gold spheres. Most of the insulin is stored in the dense cores of secretory vesicles。Farrell.) Figure 346. Western blotting or immunoblotting. The total proteins from dividing tobacco cells in culture are first separated by twodimensional polyacrylamidegel electrophoresis as shown in and their positions revealed by a sensitive protein stain (A). The separated proteins on an identical gel were then transferred to a sheet of nitrocellulose and incubated with an antibody that recognizes those proteins that, during mitosis, are phosphorylated on threonine residues. The positions of the dozen or so proteins that are recognized by this antibody are revealed by an enzymelinked second antibody (B). (From . Traas et al., Plant Journal2:723732) E. Western blotting or immunoblotting Table 48 Some Reagents Commonly Used to Cleave Peptide Bonds in Proteins Amino Acid 1 Amino Acid 2 Enzyme Trypsin Lys or Arg any Chymorypsin Phe, Trp, or Tyr any V8 protease Glu any Chemical Cyanogen bromide Met any 2Nitro5thiocyanobenzoate any Cys Figure 347. Production of a peptide map, or fingerprint, of a protein. Here, the protein was digested with trypsin to generate a mixture of polypeptide fragments, which was then fractionated in two dimensions by electrophoresis and partition chromatography. The pattern of spots obtained is diagnostic of the protein analyzed. Figure 348. Xray crystallography. (A) Protein crystal of ribulose bisphosphate carboxylase, an enzyme that plays a central role in CO2 fixation during photosynthesis. (B) Xray diffraction pattern obtained from the crystal. (C) Simplified model of the protein structure derived from the xray diffraction data. (A, courtesy of C. Branden。 they are, in reality, only onetenth this diameter. (Courtesy of P. Sammeh and G. Borisy.) Figure 359. Methods to introduce a membraneimpermeant substance into a cell. (A) the substance is injected through a micropipette. (B) the cell membrane is made transiently permeable to the substance by disrupting the membrane structure with a brief but intense electric shock. (C) membranebounded vesicles are loaded with the desired substance and then induced to fuse with the target cells. Figure 364. Indirect immunocytochemistry. The method is very sensitive because the primary antibody is itself recognized by many molecules of the secondary antibody. The secondary antibody is covalently coupled to a marker molecule that makes it readily detectable. Commonly used marker molecules include fluorescein or rhodamine dyes, the enzyme horseradish peroxidase or colloidal gold spheres, and the enzymes alkaline phosphatase or peroxidase. Figure 365. Preparation of hybridomas that secrete monoclonal antibodies against a particular antigen (X). The selective growth medium used contains an inhibitor (aminopterin) that blocks the normal biosynthetic pathways by which nucleotides are made. The cells must therefore use a bypass pathway to synthesize their nucleic acids, and this pathway is defective in the mutant cell line to which the normal B lymphocytes are fused. Because neither cell type used for the initial fusion can grow on its own, only the hybrid cells survive. 7. Monoclonal Antibodies 8. Gene Knockout mice Mario Capecchi (Late 1980s) (University of Utah) embryonic stem cells in inner cell mass as target cells 1/104 cells undergo a process of homologous rebination. 9. The technique for the take apart and gather up of cell, and microscope manipulation ?Preparation and reform of karyoplast and cytoplast ?Transgenic animals and plants Transgenic mice 10 weeks 44g and 29g THANKS! 。 C, adapted from original provided by B. Furugren.) 5. Protein structure A. Xray crystallography Figure 349. NMR spectroscopy. (A) An example of the data from an NMR machine. This is a twodimensional NMR spectrum derived from the carboxylterminal domain of the enzyme cellulase. The spots represent interactions between hydrogen atoms that are near neighbors in the protein and hence their distance apart. Complex puting methods, in conjunction with the known amino acid sequence, enable possible patible structures to be derived. In (B) 10 structures, which all satisfy the distance constraints equally well, are shown superimposed on one another, giving a good indication of the probable threedimensional structure. (Courtesy of P. Kraulis.) B. NMR spectroscopy Figure 720. In situ hybridization for RNA localization in tissues. Autoradiograph of a section of a very young Drosophila embryo that has been subjected to in situ hybridization using a radioactive DNA probe plementary to a gene involved in segment development. The probe has hybridized to RNA in the embryo, and the pattern of autoradiographic silver grains reveals that the RNA made by the gene (ftz) is localized in alternating stripes across the embryo that are three or four cells wide. At this stage of development (cellular bla