m7 - Lab#2 Proteins You compared different proteins You...

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Lab #2: Proteins You compared different proteins You separated different sized proteins using SDS-page gel electrophoresis You determined an unknown from known proteins that you have run out on the gel SDS-page: Ch3, Pages 87-89
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• Electrophoresis is a technique for separating molecules (i.e. proteins, nucleic acids) that are mixed together under the influence of an applied electrical field. • Dissolved molecules in an electric field will move, or migrate, based upon their charge:mass ratio. Solution Positive (+) Electrode Negative ( - ) Electrode -2 molecules dissolved in a liquid solution with the same size and shape, but with different net charges Electrophoresis - - - - - - - - - - - - - - - - - - - - Greater net charge will move faster
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• Many proteins that differ in size and shape have nearly identical charge:mass ratios… Gel Electrophoresis -Would electrophoresis of proteins that differ in size/shape but have identical charge:mass ratios in liquid solution result in separation of the molecules? • You can successfully separate proteins with electrophoresis in various gels (semisolid suspensions in water) rather than in liquid solution. - Polyacrylamide gels (Pore size can be varied by adjusting the concentration of polyacrylamide). -When a mixture of proteins is loaded unto a gel and an electric current is applied through the gel… • The rate at which a protein moves through a gel is influenced by the gel’s pore size and the strength of the electric field.
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Depending on the type of gel that is run: What can an electrophoresis gel tell us about a protein?
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SDS-Page • The most powerful technique for resolving protein mixtures. • Proteins are exposed to the ionic detergent SDS (sodium dodecylsulfate) before and during electrophoresis. • SDS is an ionic detergent that is able to denature proteins (disrupts weak non- covalent interactions) β -mecaptoethanol is added to protein samples …to break/reduce ???? bonds
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SDS-page: Denaturing Gel Extended conformations with similar charge:mass ratios • SDS denatures proteins, causing multimeric proteins to dissociate into their subunits • SDS coats the all polypeptide chains with a negative charge therefore forcing the polypeptide chains into extended conformation with similar charge:mass ratios. • Therefore, SDS eliminates the effect of differences in shape, and so chain length (mass) is the sole determinant of the migration rate in SDS-gel electrophoresis .
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Proteins can be run on SDS gels • The buffer in the upper and lower chamber is what helps carry the current and the protein through the gel matrix. • Notice that the proteins are going to migrate towards the bottom of the gel, does that mean that all proteins are (+)?
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• The higher the molecular weight the “slower” it migrates on the gel.
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