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Biochem Exam 1

Purification Methods

TermDefinition
Differential centrifugation Uses different rates of spinning to cause different things to go to the bottom of the centrifuge
Dialysis Separates molecules based on size by placing mixture in a bag with small enough holes for the smaller molecules (salt) to diffuse through and leave the big proteins remaining
Gel filtration/molecular exclusion Separates molecules based on size by packing tube with beads that have small holes and tunnels through which only smaller molecules can pass through; the bigger ones bypass the beads and come out first
Ion-exchange chromatography Separates molecules based on charges; solution passed through tube that has beads with charges on their surfaces. Cation exchange means the beads are negatively charged and thus the positively charged molecules will bind
Affinity chromatography Separates based on affinity for certain molecules. Protein that binds to a certain molecule will bind to bead which has molecule on surface; others will pass through. Add more molecules to wash the proteins off
High Performance Liquid Chromatography (HPLC) Separates proteins based on polarity. Reverse phase chromatography uses little beads with nonpolar molecules on surface; solution's applied at high pressure to pass them through. Most polar will come off first; nonpolar will interact with other nonpolar
Agarose gel electrophoresis Polysaccharide gel that forms large holes through which DNA and RNA pass. Electrical current so neg. at top and pos. at bottom; small will go through faster than large
Polyacrylamide Gel Electrophoresis (PAGE) Like agarose, but smaller; used for proteins instead of DNA. Have to convert globular proteins to rod-like structure; use detergent (SDS) to denature and coat proteins into rods. Longer = more negatively charged
Isoelectric focusing Separates by pI. Negative and positive electrodes on either end; most positive proteins will be closest to the negative electrode and vice versa
Two-dimensional gel electrophoresis Combination of SDS-PAGE and isoelectric focusing; separates by pI and then in each category of pI, separates by size. Every spot corresponds to a unique protein
Created by: 100000033006215