CB4351_01_S09_C05

CB4351_01_S09_C05 - Analysis of Proteins and...

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Analysis of Proteins and Macromolecules: Electrophoresis There are many methods for separating, identifying, and characterizing proteins and other macromolecules. Electrophoresis : one of the most common methods used to study proteins and other macromolecules ( e.g. DNA , RNA , etc. ) Based on the motion of charged particles in an electric field toward an electrode of opposite charge. Macromolecules differ in their electrophoretic mobility depending on their net charge , shape , and size . • Thus, electrophoresis can be used to measure net charge , shape , or size . • In addition, electrophoresis can be used to separate mixtures of macromolecules if they have different net charges, shapes, or sizes. • Of these, the most commom type of electrophoresis of proteins is separation by size.
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Skematic of a typical Electrophoresis Apparatus Cathode (attracts cations) Anode (attracts anions) support media ( buffered at a specific pH ) electrodes sample wells ( with marker dye ) to power supply to power supply
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Running a typical Protein Electrophoresis Separation Cathode (attracts cations) Anode (attracts anions) Cathode (attracts cations) Anode (attracts anions) Beginning of separation, protein samples loaded End of separation, before dying proteins (system buffered at pH 7.0)
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Running a typical Electrophoresis Separation Beginning of separation, protein samples loaded End of separation, after dying proteins ("Coomasie Blue", Silver staining, etc. ) Cathode (attracts cations) Anode (attracts anions) Cathode (attracts cations) Anode (attracts anions) (system buffered at pH 7.0) Question: What would happen if the experiment was run for a very long time?
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Analysis of a typical Electrophoresis Separation Questions: What is the minimum number of different proteins in each lane? Is there any evidence that some of the same proteins are in each lane? (Does this evidence definitively "prove" that there are the same proteins in some lanes?) Which lane contains "acidic" proteins only? (e.g. proteins with a high content of acidic residues) Which lane contains "basic" proteins only? (e.g. proteins with a high content of
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CB4351_01_S09_C05 - Analysis of Proteins and...

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