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Protein complexes
Uni of Notts, Structure, function, & analysis of Proteins, year 2, topics 18-20
| Term | Definition |
|---|---|
| Protein complex | A set of proteins that form stable or transient functional non-covalent associations, often with dynamic or signal-induced interactions |
| Protein domain | Compact functional/structural region within a protein that can fold independently & often mediates interactions |
| Colocalisation | 2 proteins within the same spatial compartment of the cell |
| Difference between protein colocalisation & interaction | Often colocalised proteins directly interact but they can be in the same compartment by coincidence or indirectly interact; direct binding still has to be proven |
| Multisubunit protein complex *example* | The 26S proteasome is a multisubunit protease complex |
| Interactome | Full compliment of molecular interactions in a cell, especially protein–protein interactions |
| Widespread effects of interactome dysfunction | One altered interaction can disrupt multiple downstream pathways & complexes causing widespread dysfunction |
| Indirect immunohistochemistry | Uses a primary antibody plus enzyme-linked secondary antibody to produce a colourimetric signal after fixation |
| Direct immunohistochemistry | Binding the antigen of interest with a primary antibody linked to an enzyme or other tag to produce a colourimetric signal |
| Advantage of indirect over direct labelling | Indirect methods amplify signal because multiple secondary antibodies can bind each primary antibody |
| Direct & indirect immunofluorescence | Localises endogenous proteins in fixed cells in cultures (e.g., on a coverslip), using fluorescent tags instead of enzymes |
| Experimental colocalisation assesment | Tag each protein separately, merge the signals, & compare overlap in the same cells/structures |
| Immunogold EM | EM localisation method using gold nanoparticle-conjugated antibodies; gold is electron dense & visible in the microscope |
| Yeast two-hybrid | Splits DNA-binding & activation domains of TFs involved in growth on specific media with each domain at the C & N-termini. If the yeast grows, then the domains interacted & assembled the TF |
| Immunoprecipitation | Using antibodies to precipitate the protein of interest out of solution, often with its binding partners |
| LC-MS/MS | Liquid chromatography separates peptide mixtures before tandem mass spectrometry fragments & identifies them. Also known as shotgun-proteomics |
| How LC separates peptides | Using hydrophobicity on a reverse-phase column rather than sequence order |
| Benefits of shotgun proteomics | Identify thousands of proteins from complex mixtures in a high-throughput, untargeted way |
| How the database identifies proteins from tryptic peptides | Matches peptide spectra to theoretical peptide fragments from reference sequences to align full sequences |
| Why you don't need the whole protein sequence for identification | A few confidently matched tryptic peptides are enough to identify the protein uniquely |
Created by:
Denny12
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