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6 Cards in this Set
- Front
- Back
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FRET |
Controls: Test interaction of fluorophore with non-binding partner, confirm protein expression, cells only express individual proteins Advantages: Low-affinity interactions, identify cell compartment, in-cell testing, RT Disadvantages: Fluorophore interference, photobleaching, indirect binding could yield no results |
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BIFC |
Controls: Negative control (proteins w/o fluorescent fragments, fluorescent fragments alone), confirm fusion with fragment Advantages: Detects weak/transient interactions, stabilizes them, direct visualization, RT in-vivo analysis Disadvantages: Steric hindrance possible, delay between interaction and fluorescence, cellular compartment |
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Far-Western |
Controls: Purified protein WITHOUT label, secondary antibody only Advantages: Confirms direct interaction, determines MW, identifies direct binding partner Disadvantage: Identity of binding partner not known, nitrocellulose interference, irrelevant with compartments |
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Yeast Two-Hybrid |
Controls: Co-transfect without presence of second reporter (Trp/Leu/His), empty plasmid, re-transform new cells w/plasmids, check for frame of candidate interacting construct, many others Advantages: Strength of interaction not important, good for testing in-vivo, screen multiple partners, test specific interaction Disadvantages: drawn-out process, compartment reason, interaction may not happen if chimera with domain |
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Co-IP |
Controls: Confirm IP of protein of interest antibody should only be specific for one protein, nonspecific IgG binding control Advantages: Rapid, tests for interaction of proteins in vitro or in cell Disadvantages: Does not identify compartment or interaction specificity. Must be stable and high strength |
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Pull-Down Assay |
Control: Test GST-protein alone, ensure GST fusion, use only GSH agarose beads. Advantage: Rapid, no need for antibody, show domain interactions Disadvantages: Compartment, interaction specificity not known, GST-fusion may interfere via N-terminal |
