Experimental Evidence Codes
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experimental_systems [2018/10/06 09:13] biogridadmin [Experimental Evidence Codes] |
experimental_systems [2024/06/07 14:09] (current) rose [Physical Interactions] |
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| * **Co-purification** - An interaction is inferred from the identification of two or more protein subunits in a purified protein complex, as obtained by several classical biochemical fractionation steps, or else by affinity purification and one or more additional fractionation steps. Note that a Western or mass-spec may also be used to identify the subunits, but that this differs from "Affinity Capture-Western" or "Affinity Capture-Mass Spec" because it involves at least one extra purification step to get rid of contaminants (e.g. [[https://www.ncbi.nlm.nih.gov/pubmed/19343713?dopt=Abstract|PMID: 19343713]]). Typically, TAP-tag experiments are considered to be affinity captures and not co-purification experiments. If there is no obvious bait-hit directionality to the interaction, then the co-purifying proteins should be listed as a complex. If only co-fractionation is demonstrated, i.e. if the interaction is inferred from the presence of two or more protein subunits in a partially purified protein preparation (e.g. [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11294905&query_hl=4&itool=pubmed_DocSum|PMID: 11294905]], Fig. 9), then use "Co-fractionation" instead. | * **Co-purification** - An interaction is inferred from the identification of two or more protein subunits in a purified protein complex, as obtained by several classical biochemical fractionation steps, or else by affinity purification and one or more additional fractionation steps. Note that a Western or mass-spec may also be used to identify the subunits, but that this differs from "Affinity Capture-Western" or "Affinity Capture-Mass Spec" because it involves at least one extra purification step to get rid of contaminants (e.g. [[https://www.ncbi.nlm.nih.gov/pubmed/19343713?dopt=Abstract|PMID: 19343713]]). Typically, TAP-tag experiments are considered to be affinity captures and not co-purification experiments. If there is no obvious bait-hit directionality to the interaction, then the co-purifying proteins should be listed as a complex. If only co-fractionation is demonstrated, i.e. if the interaction is inferred from the presence of two or more protein subunits in a partially purified protein preparation (e.g. [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11294905&query_hl=4&itool=pubmed_DocSum|PMID: 11294905]], Fig. 9), then use "Co-fractionation" instead. | ||
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| + | * **Cross-Linking-MS (XL-MS)** - An interaction is detected between two proteins using chemically reactive or photo-activatible cross-linking reagents that covalently link amino acids in close proximity, followed by mass spectrometry analysis to identify the linked peptides (reviewed in [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=37406423&query_hl=4&itool=pubmed_DocSum|PMID: 37406423]], [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=37104977&query_hl=4&itool=pubmed_DocSum|PMID: 37104977]]). Experiments may be carried out with live cells or cell lysates in which all proteins are expressed at endogenous levels (e.g. [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=34349018&query_hl=4&itool=pubmed_DocSum|PMID: 34349018]], [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=35235311&query_hl=4&itool=pubmed_DocSum|PMID: 35235311]]) or with recombinant proteins (e.g. [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=28537071&query_hl=4&itool=pubmed_DocSum|PMID: 28537071]]). | ||
| * **Far Western** - An interaction is inferred when a bait protein is immobilized on a membrane and a prey protein that is incubated with the membrane localizes to the same membrane position as the bait protein. The prey protein could be provided as a purified protein probe (e.g. [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12857883&query_hl=4&itool=pubmed_DocSum|PMID: 12857883]], Fig. 7). | * **Far Western** - An interaction is inferred when a bait protein is immobilized on a membrane and a prey protein that is incubated with the membrane localizes to the same membrane position as the bait protein. The prey protein could be provided as a purified protein probe (e.g. [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12857883&query_hl=4&itool=pubmed_DocSum|PMID: 12857883]], Fig. 7). | ||