Movement of protein from nucleus to cytoplasm can he seen by:
Ans. B. FRAP Movement of protein from nucleus to cytoplasm can be seen by Fluorescence Recovery After Photobleach ing (FRAP)."FRAP is based on the principal of observing the rate of recovery of fluorescence in a photobleached area of cell, due to the movement of a fluorescent marker into the area. The method is to tag a specific cell component (i.e. protein, lipid, carbohydrate) with afluorescent molecule; image that cell, photobleach a small portion of the cell, and then image the recovery offluorescence over time. The recovery of fluorescence in the photobleached portion occurs because of diffusion or active movement of fluorophore tagged molecules within the cell replacing the bleached fluorophore with unbleached molecules. FRAP is used to investigate the diffusion and motion of biological macromolecules, including movement into and out of the nucleus, and lateral movement within membranes. FRA P has been used to assess the structure of artificial and biological membranes. FRA P was used to help define the fluid mosaic model of cell membranes.Fluorescence Recovery After Photobleaching (FRAP)* Lateral diffusion can be shown experimentally by attaching fluorescent probes to the head groups of lipids.' proteins and using the fluorescence microscopy to follow the probes over time.* Two techniques involve:Fluorescence Tagging of Membrane SurfaceSingle Particle Tracking* Lipids {protein etc) on the outer leaflet (layer) of membrane are labeled by reaction with a membrane -impermanent fluorescent probe, so the surface is uniformly labeled when viewed with fluorescence microscope. A small area (5mm2) of a cell surface with fluorescent tagged lipids is photobleached by intense laser radiation so that the irradiated patch no longer fluoresces (emit fluorescent light) when viewed with less intense (non bleaching) light in the florescence microscope.* However the photo bleached membrane region recovers its fluorescence within milliseconds as unbleached lipid molecules diffused into the bleached patch and bleached lipid molecules diffuse away from it by rapid lateral diffusion.* The rate of fluorescence recovery after photo bleaching or FRAP is a measure of rate of lateral diffusion of the lipids* FRAP can be used to assess structure of membranes (biological and artificial both), define fluid mosaic model and investigate diffusion (motion) of biological macromolecules including lateral diffusion movements with in the plane of membranes and movements into and out of nucleus.* The motion (movement) of a single fluorescently labeled lipid (protein / etc) molecule in the cell membrane is recorded on video by fluorescence microscopy on a much shorter time scale (eg 50 ms = 2250 frames with time resolution of 25ps = 40000 frames/'s).* The pattern of movement confirms rapid lateral diffusion within a confined (small discrete) region with occasional hops into an adjoining region. It suggests that lipids are corralled by molecular fences that they can occasionally jump by hop diffusion.Membrane proferns (like glycophorin and Cl- HCO2- exchanger in RBC) immobilized by their association with spectrin (a filamentous cytoskeleton protein) form the fences that define the regions of relatively unrestricted lipid motion.Hop Diffusion of Single Lipid Molecule
FISH
FRAP
Confocal microscopy
Electron microscopy
Miscellaneous (Structure & Function of Proteins)