06_PAGE_103009 - Polyacrylamide Gel Electrophoresis (PAGE)

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Polyacrylamide Gel Electrophoresis (PAGE) LABORATORY EXERCISE #6 Rev: 3/10/10 Materials and Methods Polyacrylamide Gel Electrophoresis   was used to analyze whole-cell lysates and extracellular protein production. Extracellular proteins were obtained by growing bacteria overnight at 37 o C on a dialysis membrane (Spectra/Por 12-14,000 MWCO; Spectrum Medical Industries, Huston TX) overlaid onto a TSA plate. Cells and extracellular proteins were isolated by adding 100 µl of Tris-buffered saline (pH 7.2) to the membranes and scraping the growth into a microcentrifuge tube; the bacteria were separated from the extracellular proteins by centrifugation. Protein concentration was determined colormetrically. Approximately 10 µl of packed, washed cells or 10 µg of protein were  boiled for three minutes in reducing buffer and were applied to an individual lane of a 4- 15% polyacrylamide gel (Ready Gel; Bio-Rad, Hercules CA).   Electrophoresis is the movement of charged molecules through an electric field; it is one method used to resolve different protein antigens in a complex mixture Protein Electrophoresis - When voltage is applied along the gel, proteins migrate into it at different speeds (different electrophoretic mobilities) separating into bands within each lane - Most proteins are charged molecules and will exhibit electrophoretic migration dependent on the pH of the buffer through which the proteins travel - In acid solutions, the amino groups of proteins are positively charged while the carboxyl groups are not charged - In a basic or alkaline solution, the carboxyl groups are negatively charged while the amino groups are not charged … Therefore, in the alkaline buffers commonly used for electrophoresisn most proteins have a net negative charge and will migrate toward the anode (+ electrode) - The buffer used for electrophoresis has a moderately high ionic strength to permit rapid protein migration resulting in sharper protein migration zones - But very high ionic strength buffers can cause too much heat production during electrophoresis, which leads to distortion of the protein bands … Some electrophoresis units are equipped with sophisticated cooling mechanisms to help overcome this problem - At the same time, the buffer components assume a positive charge and move toward cathode (- electrode) in a process known as electroosmosis* * Some proteins, such as gamma globulin, are too weakly charged to overcome the electroosmotic forces and will actually be "back-washed" toward the cathode in agarose gels - Smaller proteins migrate faster through this mesh and the proteins are thus separated according to size (usually measured in kilo Daltons, kD) 1
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Electrophoresis - Electrophoretic separation of proteins by molecular size requires SDS treatment and denaturation of the proteins … … Sodium dodecyl sulfate (SDS ) is an anionic detergent which binds to the protein and
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This note was uploaded on 03/09/2010 for the course BIOL 371 taught by Professor Eugenemuller during the Fall '09 term at Framingham State College.

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06_PAGE_103009 - Polyacrylamide Gel Electrophoresis (PAGE)

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