课件-国外大学细胞生物学课件-英文5.bioy.ppt

How is immunofluorescence done in a lab? W2011 D103 - lecture 5 @ Suetterlin and MacGregor - all rights reserved. * Fluorescent light excitation emission Light Nucleus microtubules Golgi Primary antibody (Rabbit anti tubulin Ab) recognizes antigen (eg tubulin) Secondary antibody (Goat anti-rabbit Ab) recognizes the constant region of rabbit antibodies Recognizes Golgi protein Fluorescent light Light more than one secondary antibody binds to one primary antibody ? signal enhancement antibody coupling is inefficient - precious primary antibody would be lost during the coupling process. flexibility in the the color of the fluorochrome - combinatorial stainings with Green or Red fluorescent proteins Why are secondary antibodies used? W2011 D103 - lecture 5 @ Suetterlin and MacGregor - all rights reserved. * Important concepts, lecture 5 W2011 D103 - lecture 5 @ Suetterlin and MacGregor - all rights reserved. * The cytoskeleton gives a cell mechanical support, but is dynamic. 3 main cytoskeletal structures: - actin filaments and microtubules ? assemble into polar structures in an energy-dependent - intermediate filaments ? apolar structures whose assembly is energy-independent - each of these cytoskeletal elements form higher order structures Characteristics of actin filaments (F-actin): Assembly from ATP-bound G-actin ? polar filament (6 nm in diameter) Nucleation is the rate limiting step Undergoes treadmilling at cytosolic ATP-G-actin concentration that are higher than the critical concentration at the (+) end, and that are lower than the critical concentration at the (-) end (Cc- Cc cyto Cc+) The cytosolic concentration of ATP-bound G-actin is tightly regulated Capping proteins regulate the dynamics of actin filaments * * * * * * * * * * * * * * * * * * * Lecture 5The cytoskeleton: general principles W2011 D103 - lecture 5 @ Suetterlin and MacGregor - all rights reserved. * Introduction Actin filaments and their characteristics W2011 D103 - lec