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Biochemistry

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Purines, Pyrimidines and Nucleic Acid Metabolism

Question
14 out of 59
 

Which of the following method is use for detection of protein motion from cell nuclei to cytoplasm(AIIMS

May 2010)



A FISH
B FRAP

C Electron microscopy
D Confocal microscopy

Ans. B FRAP

a. Fluorescence recovery after photobleaching (FRAP) denotes an optical technique capable of quantifying the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells.

b. This technique is very useful in biological studies of cell membrane diffusion and protein binding. In addition, surface deposition of a fluorescing phospholipid bilayer (or monolayer) allows the characterization of hydrophilic (or hydrophobic) surfaces in terms of surface structure and free energy.

c. Similar, though less well known, techniques have been developed to investigate the 3-dimensional diffusion and binding of molecules inside the cell; they are also referred to as FRAP.

d. This technique is commonly used in conjunction with green fluorescent protein (GFP) fusion proteins, where the studied protein is fused to a GFP. When excited by a specific wavelength of light, the protein will fluoresce. When the protein that is being studied is produced with the GFP, then the fluorescence can be tracked.

e. Photodestroying the GFP, and then watching the repopulation into the bleached area can reveal information about protein interaction partners, organelle continuity and protein trafficking.

f. If after some time the fluorescence doesn't reach the initial level anymore, then some part of the fluorescence is caused by an immobile fraction (that cannot be replenished by diffusion). Similarly, if the fluorescent proteins bind to static cell receptors, the rate of recovery will be retarded by a factor related to the association and disassociation coefficients of binding.

g. This observation has most recently been exploited to investigate protein binding.

Purines, Pyrimidines and Nucleic Acid Metabolism Flashcard List

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