DNA lacZ lacI O lacY lacA C E P rep T CAP repressor CAP site pol-binding site operator structural genes terminator lac operon lactose cAMP 8.

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Presentation transcript:

DNA lacZ lacI O lacY lacA C E P rep T CAP repressor CAP site pol-binding site operator structural genes terminator lac operon lactose cAMP 8

DNA lacZ lacI O lacY lacA C E P rep galactose indoxyl T CAP repressor CAP site pol-binding site operator structural genes terminator IPTG X-Gal - X-Gal chromogenic substrate lacZ gene 9

DNS DNA lacZ lacI O lacY lacA C E P trans- acetylase permease rep  -gal T CAP pol repressor CAP site pol-binding site operator structural genes terminator lacZ gene - X-Gal chromogenic substrate 9

DNA lacZ lacI O lacY lacA C E P T lacZ gene in cloning lacZ  -fragment   - fragment  lacZ  -  Foreign DNA repressor CAP site pol-binding site operator structural genes terminator 10

7. Immunohistochemistry Y Y Y YY Y nucleus cytoplasm Cell surface antigenes anti-C antibody Fluorescent dye Rabbit anti-C Primary antibody Goat anti-rabbit Secondary antibody Direct methodIndirect method B A A B B B B B A A A A C C C C - immunofluorescence Citoplasmic antigenes cytoplasm D E Cytoplasmic antigenes D E Cell surface antigens 27

peroxidase Y Y Y Y nucleus cytoplasm Cell surfice antigens peroxidase Rabbit anti-C primary antibody Goat anti-rabbit secondary antibody Indirect methods B B B A A A C C Y peroxidase Y avidin - peroxidase - biotin – avidin – peroxidase Anti-rabbit antibody (secondary antibody) - immunoperoxidase-based methods DAB + H 2 O 2 Ni-DAB + H 2 O 2 cytoplazmic antigens anti-rabbit antibody (secondary antibody) D E Y anti-C antibody Y 7. Immunohistochemistry 28 biotin

avidin ABC method (ABC = avidin-biotin complex) anti-rabbit antibody (secondary antibody) Y peroxidase Y primary antibody antigen 7. Immunohistochemistry - immunoperoxidase-based methods 29 DAB + H 2 O 2 Ni-DAB + H 2 O 2 Y Y

Y Y Y Y nucleus cytoplasm Alkaline phosphatase Rabbit anti-C primary antibody Goat anti-rabbit secondary antibody Indirect method B B B A A A C C - alkaline phosphatase - digoxigenin – anti-digoxigenin – alkaline phosphatase alk. phosph. Y Sheep anti-rabbit antibody alk. phosph Y Y DIG Anti-DIG antibody anti-C antibody Cell surfice antigens cytoplasmic antigens D E Y Rabbit anti-C antibody 30 Y Y alk. phosph DIG Anti-DIG antibody anti-C antitbody Y anti-rabbit antibody 7. Immunohistochemistry

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