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Study of Diffusion in Starch Systems by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0032-WXH)

Introduction

Starch is composed of two high molecular weight polymers of glucose: linear amylose and the highly nonrandomly branched and semicrystalline amylopectin. The most commonly encountered physical form of starch when in conventional use is the gelatinized starch paste, which is made up of disrupted granules in a matrix of amylose and amylopectin that gels and eventually retrogrades and crystallizes upon storage. <br />In all cases, diffusion of the constituent polymer chains and the resulting system viscosity (and especially the way in which this changes as the result of various processing techniques or physical transformations/storage conditions) and the diffusion of smaller trace components and added material (sugars, flavor compounds, enzymes, etc.) are of great significance with regard to the material properties of the starch.




Principle

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique capable of quantifying the mobility of molecules within cells. By exploiting the phenomenon of photobleaching, fluorescent mole- cules within a region of interest can be selectively and irreversibly 'turned off'. It is capable of quantifying the two-dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells.

Applications

• Characterization of the mobility of individual lipid molecules within a cell membrane.
• Analysis of molecule diffusion within the cell
• Study of protein interaction partners, organelle continuity and protein trafficking.

Procedure

1. An initial fluorescence of fluorescent molecules is measured in the region of interest (ROI).
2. The fluorescent molecules are rapidly photobleached by focusing the high-intensity laser beam onto the defined area.
3. The exchange of bleached molecules with unbleached molecules from the surrounding region is followed over time using a low-intensity laser.

Materials

• Optical microscope.
• Light source.
• Fluorescent probe.