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--- experimental_systems [2018/10/06 09:13]
biogridadmin [Experimental Evidence Codes]
+++ experimental_systems [2025/07/28 12:50] (current)
jenn [Physical Interactions]
@@ Line 25: / Line 25: @@
   *  **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.
+  *  **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).
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   * **Proximity Label-MS** - An interaction is inferred when a bait-enzyme fusion protein selectively modifies a vicinal protein with a diffusible reactive product, followed by affinity capture of the modified protein and identification by mass spectrometric methods, such as the BioID system [[http://www.ncbi.nlm.nih.gov/pubmed/?term=24255178|PMID: 24255178]]. This system should not be used for //in situ// proximity ligation assays in which the interaction is measured by fluorescence, eg. [[http://www.ncbi.nlm.nih.gov/pubmed/25168242?dopt=Abstract|PMID: 25168242]], which should be captured as co-localization.
-  *  **Reconstituted Complex** - An interaction is inferred between proteins //in vitro//. This can include proteins in recombinant form or proteins isolated directly from cells with recombinant or purified bait. For example, GST pull-down assays where a GST-tagged protein is first isolated and then used to fish interactors from cell lysates are considered reconstituted complexes (e.g. [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14657240&query_hl=4&itool=pubmed_DocSum|PMID: 14657240]], Fig. 4A or [[http://www.ncbi.nlm.nih.gov/pubmed/?term=14761940|PMID: 14761940]], Fig. 5). This can also include gel-shifts and surface plasmon resonance experiments. The bait-hit directionality may not be clear for 2 interacting proteins. In these cases the directionality is up to the discretion of the curator.
+  *  **Reconstituted Complex** - An interaction is inferred between proteins //in vitro//. This can include proteins in recombinant form or proteins isolated directly from cells with recombinant or purified bait. For example, GST pull-down assays where a GST-tagged protein is first isolated and then used to fish interactors from cell lysates are considered reconstituted complexes (e.g. [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14657240&query_hl=4&itool=pubmed_DocSum|PMID: 14657240]], Fig. 4A or [[http://www.ncbi.nlm.nih.gov/pubmed/?term=14761940|PMID: 14761940]], Fig. 5). This can also include gel-shifts, surface plasmon resonance experiments and bio-layer interferometry (BLI) experiments. The bait-hit directionality may not be clear for 2 interacting proteins. In these cases the directionality is up to the discretion of the curator.
   *  **Two-hybrid** - An interaction is inferred when a bait protein is expressed as a DNA binding domain (DBD) fusion, a prey protein is expressed as a transcriptional activation domain (TAD) fusion and the interaction is measured by reporter gene activation (e.g. [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=9082982&query_hl=4&itool=pubmed_DocSum|PMID: 9082982]], Table 1).
+  *  **Surface Display** - An interaction is inferred when a protein of interest is fused to a surface protein and assayed for interaction with another protein or ligand. The surface fusion is often expressed in yeast and termed yeast display [[https://pubmed.ncbi.nlm.nih.gov/39322662/|PMID: 39322662]], but can also be expressed in bacteriophage and termed phage display [[https://pubmed.ncbi.nlm.nih.gov/10585717/|PMID: 10585717]], or other microbes and termed bacterial surface display [[https://pubmed.ncbi.nlm.nih.gov/21146237/|PMID: 21146237]] other expression systems. The displayed protein is then used to screen potential binding partners (such as antibodies, peptides, or other proteins). Binding can then be detected by the presence or cellular uptake ([[https://pubmed.ncbi.nlm.nih.gov/39322662/|PMID: 39322662]], Fig. 1C) of a fluorescent reporter.
+  *  **Thermal Shift Assay** - An interaction is demonstrated between a protein and a ligand by measuring the change in a protein's thermal stability upon ligand binding. This shift can be measured by nano-differential scanning fluorimetry (Nano-DSF) (e.g.[[https://pubmed.ncbi.nlm.nih.gov/36650907/|PMID: 36650907]], Fig. 2 and 3), differential scanning calorimetry (DSC) (e.g. [[https://pubmed.ncbi.nlm.nih.gov/32828280/|PMID: 32828280]], Fig. 1D) or other methods.
 ===== Genetic Interactions =====