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Protein purification
Uni of Notts, Structure Function & Analysis of Proteins, year 2, topic 4
| Term | Definition |
|---|---|
| Column chromatography | Separates molecules based on physical or chemical properties by adsorbing them using various components to alter the flow-through speed of different molecule types based on these properties |
| Using gel filtration/size‑exclusion chromatography to separate proteins | Filled with porous polymer beads. Large proteins elute first because they cannot enter porous beads; small molecules enter pores & elute later |
| Detecting protein yield & purity in column fractions using absorbance & graphs | Peptide bonds absorb 220nm, aromatic residues 280, & nucleic acid contaminants 260. These can be plotted on a curve of the absorbance spectrum, area under curve = purity or 260:280 ratio |
| What the number of peaks in a chromatogram indicates | Fewer peaks indicate higher purity of protein of interest; multiple peaks indicate mixed proteins |
| Cation‑exchange chromatography | Negatively charged beads bind positively charged (basic) proteins, which are eluted by increasing salt concentration into multiple fractions ranging in protein basicity |
| IMAC & uses | Immobilised Metal Affinity Chromatography uses metal ions bound to beads (e.g., Ni²⁺) to bind engineered tags like His‑tags |
| Affinity chromatography | Specific ligand on beads to bind protein of interest based on known interactions. Often uses known protein-protein interactions between domains |
| How 1D SDS-PAGE separates proteins | Molecular weight after denaturation & uniform negative charge density coating as well as a constant mass:charge ratio |
| Why β‑mercaptoethanol is used in SDS‑PAGE | Reduces disulfide bonds to sulfhydryl, preventing cross‑linking that would distort migration |
| Chaotropes | A chemical that disrupts non‑covalent interactions, helping denature proteins & membranes |
| How acrylamide percentage affect SDS‑PAGE | Higher % acrylamide creates a tighter matrix for resolving smaller proteins; lower % resolves larger proteins |
| 2D electrophoresis | Separates proteins by isoelectric point (pI) using differently charged chemicals horizontally & molecular weight vertically |
| Why 2D electrophoresis produces spots instead of bands | Proteins are separated by molecular weight & pI, increasing resolution so each spot likely represents a single protein. Many proteins have similar mass but few have similar mass & pI |
| What spot intensity represents in 2D gels | Protein abundance |
| How proteomic analysis of 2D gels is used to compare conditions | Overlaying spot patterns between conditions of an experiment demonstrates a causal relationship between the dependant variables & the expression of certain proteins |
| Combining purification steps & running them on an SDS-PAGE | Each purification protocol removes specific contaminants & reduces PAGE bands compared to the lysate & previous steps until only a single band remains at which point the protein is considered pure |
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Denny12
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