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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
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
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
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
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
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
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