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Biochem Exam 1
Purification Methods
| Term | Definition |
|---|---|
| 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 |
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