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Lab 03 Part II Spring 2012

# Lab 03 Part II Spring 2012 - BSCI330 Laboratory Manual...

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BSCI330 Laboratory Manual Spring 2012 1 | P a g e LAB EXERCISE 3 Part II Protein Gel Electrophoresis and 3D modeling I. Introduction Last week, you explored the physical properties of proteins isolated from blood components through analysis of two important protein precipitation methods: salt precipitation and organic solvent precipitation. In the first part of this week’s lab, you will analyze the protein content of the various fractions you collected by carrying out a commonly used technique in cell biology laboratories: chromatographic separation of proteins through electrophoresis. The specific technique of choice for this analysis is sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) . In the second part of this week’s lab, you will further explore protein structure through the use of a computer program that simulates 3D modeling of proteins. You should already be somewhat familiar with SDS-PAGE, as this technique is carried out in BSCI105 laboratory courses. You will need to use your textbook to review this concept (see chapter 8), and keep the following major points in mind: Gel Electrophoresis Gel electrophoresis is a process in which charged molecules migrate in an electric field , permitting the separation of nucleic acids and proteins on the basis of size. Comparisons of distance migrated in this electric field with those of known proteins or nucleic acids permits the accurate determination of size for unknown samples. The theoretical basis of the electrophoretic process may be described as follows: The movement of a charged molecule in an electric field is described by the equation: E z = f v where E is the force or strength of the electric field (in volts/cm), z is the net charge on the molecule, v is the velocity at which the molecule is moving (in cm/sec), and f is a frictional coefficient, which depends on the size and shape of the molecule, such that the larger and/or more asymmetric the particle’s shape, the higher its f value. Rearranging the equation, to solve for v : v = E z or v = E z f f demonstrates that the velocity of a migrating, charged particle through an electric field is directly proportional to both the net charge on the molecule ( z ) and the strength of the field ( E ). So, the stronger the electric field, the faster the particle will move. The equation also shows us that velocity is inversely proportional to the frictional coefficient ( f ). So, the larger and/or more asymmetrical its shape, the greater the frictional force or drag, and the slower its velocity will be. Molecular weight is a significant component of the frictional coefficient, f . Because of this, the ( z / f ) term is referred to as the charge-mass ratio of the particle. Given that every protein has a unique amino acid make-up, and that sum total of charged R groups in a

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BSCI330 Laboratory Manual Spring 2012 2 | P a g e protein determines its overall net charge, z , one would expect each protein to have a unique net charge under a given set of pH conditions. Because the goal of most standard protein gel
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