Biochemistry 2280A Study Guide - Midterm Guide: Sodium Dodecyl Sulfate, Column Chromatography, Differential Centrifugation
30 Apr 2018
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Biochemistry 2280 – Midterm
Protein Purification
The purification process
- proteins are complex mixtures (different proteins, as well as some DNA, lipids, small
molecules, etc.)
- we need to remove unwanted material (contaminants)
Preparative and analytical methods
Preparative (start with this)
• homogenize (bust up) material
• large scale; divide mixture into fractions
Analytical (follow with this)
• detect specific protein of interest
• small scale; small samples of fractions for analysis
Preparative method
Step 1 — homogenize
• break open tissue or cells to release protein
• sound waves, grinders, blenders, pressure,
detergents, lysozyme
Step 2 — centrifugation
• spin homogenate to separate by size/density
• fixed arm on left; swinging arm on right
• differential centrifugation
- low speed (whole cells, nuclei, large debris)
- medium (mitochondria, lysosomes)
- high speed (ribosomes, viruses, large macromolecules)
Step 3 — column chromatography
• further separates proteins based on characteristics
(charge/size)
• requires mobile phase (aqueous buffer solution) and solid
phase (usually small beads; matrix)
• proteins in mobile phase interact (or not) with matrix
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Proteins are complex mixtures (different proteins, as well as some dna, lipids, small molecules, etc. ) We need to remove unwanted material (contaminants) Preparative (start with this: homogenize (bust up) material, large scale; divide mixture into (cid:858)fractions(cid:859) Analytical (follow with this: detect specific protein of interest, small scale; small samples of fractions for analysis. Step 1 homogenize: break open tissue or cells to release protein, sound waves, grinders, blenders, pressure, detergents, lysozyme. Step 2 centrifugation: spin homogenate to separate by size/density, fixed arm on left; swinging arm on right, differential centrifugation. Low speed (whole cells, nuclei, large debris) Ion exchange: relies on attraction of opposite charges, ve charged proteins will bind (exchanging ions w/ previously bound, to get a protein off, increase salt concentration (charges vary with ph) Gel filtration: beads contain holes, if a protein is small enough get into the holes, it is slowed down.
