NTR366L_spring2011_week10_FishproteinPAGE_instruction

NTR366L_spring2011_week10_FishproteinPAGE_instruction - Lab...

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Lab Day 1, Week of April 18, 2011 Polyacrylamide Gel Electrophoresis (PAGE) of Proteins Goal: To demonstrate denaturing PAGE of proteins from various fish Procedure: Proteins differ not only by molecular weight but also by net charge and higher-order (secondary, tertiary, and quaternary) structure. In order to separate proteins solely on the basis of molecular weight, several steps are required. To compensate for different net charges, the samples and the electrophoresis buffer all contain sodium dodecyl sulfate (SDS), a negatively charged detergent that uniformly coats the proteins with negative charge. SDS, in combination with boiling heat, also denatures the proteins, thereby disrupting higher-order structure. The protein samples are also treated with β - mercaptoethanol, a reducing agent that breaks disulfide bonds (and also smells like rotten eggs). Under these conditions, proteins migrate during electrophoresis as DNA does, at rates inversely proportional to the log 10 of their molecular weights. Agarose is typically not used for protein electrophoresis because it lacks sufficient
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This note was uploaded on 05/10/2011 for the course NTR 366L taught by Professor Zhu during the Spring '11 term at University of Texas at Austin.

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NTR366L_spring2011_week10_FishproteinPAGE_instruction - Lab...

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