03-ProteinElectrophoresis - BSCI330 Cell biology and...

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BSCI330 – Cell biology and physiology Fall 2009 LAB MANUAL – Lab Exercise #3 Carpenter et al. 2009. BSCI330 Laboratory Manual. University of Maryland, College Park. 1 EXERCISE 3: SEPARATION OF PROTEINS AND DETERMINATION OF SPECIES SUBSTITUTION In almost every situation, proteins are the functional macromolecules of cells. Since cells need to carry out many different functions, the typical cell is a complex mixture of thousands of different proteins. The first step in identifying, characterizing and studying a protein is to be able to isolate it from all of the other proteins and macromolecules of the cell. Therefore, effective techniques of protein separation are an essential component of cell biology. This experiment examines one of these separation techniques. Recall that each different protein has a unique size (molecular weight) and sequence of amino acids (primary structure) that determines the folding pattern, three-dimensional arrangement and subunit composition (secondary, tertiary and quaternary levels of structure). Investigators often take advantage of these differences or other characteristics of proteins when attempting to purify or isolate one of interest. These separation techniques are often collectively referred to as chromatography . You may well have separated plant photosynthetic pigments in high school biology using paper chromatography, or separated chemicals in organic chemistry lab using thin-layer chromatography. On the opposite end of the spectrum from these relatively simple procedures would be something like high pressure liquid chromatography (HPLC) which is commonly used in many research labs. No matter the details, however, the basic principle is usually identical. The mixture of molecules is moved within a fluid or mobile phase through an inert or immobile stationary phase . Depending upon the degree of interaction of each particular macromolecule with the stationary phase, the rate of movement through the matrix will vary. Since the particular molecular characteristic determining the interaction may vary across the population of molecules, their rates of migration through the stationary phase differ, and separation ensues. Some molecular characteristics that have been employed to separate molecules chromatographically include charge, polarity, specific affinities, as well as molecular size and weight. In this experiment, you will utilize a technique—gel electrophoresis—to separate proteins from others in a mixture and to estimate the approximate molecular weight of an isolated protein. Recall that the basic unit of molecular weight is the Dalton (atomic mass unit), which is defined as 1/12 the weight of an atom of carbon-12. Since proteins are typically large macromolecules, their molecular weights are usually expressed in kiloDalton or kD .
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This note was uploaded on 12/15/2009 for the course BSCI 330 taught by Professor Payne during the Spring '08 term at Maryland.

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03-ProteinElectrophoresis - BSCI330 Cell biology and...

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