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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).
 
experimental_systems.1538831580.txt.gz · Last modified: 2018/10/06 09:13 by biogridadmin