Experimental Details and the TRANSPATH Quality Matrix
Details about the materials and methods used to determine interactions between and among TRANSPATH molecules are curated, when the specific information is provided in the literature. Descriptions of materials and methods curated in TRANSPATH are provided in the tables below.
| Code | Source | Description |
|---|---|---|
| A1 | Wild type | These terms describe a whole organism or its parts. The terms can refer to a wild-type organism, a naturally-occurring mutant organism, or a genetically manipulated organism. |
| A2 | Naturally-occurring chromosomal mutants | |
| A3 | Knock out | |
| A4 | Transgenic organism | |
| A5 | Organism with induced but not defined mutation(s) | |
| B1 | Embryonic cells | These terms describe primary cultures of cells, possibly transfected. The terms do not describe permanent cell lines. |
| B2 | Stem cells -> pluripotent | |
| B3 | Primary cells | |
| B4 | Recombinant cells/tissue/organisms (transfected); not permanent cell lines | |
| C1 | Permanent tissue culture cells | Permanent cell lines (permanent cultures of cells). |
| D1 | Permanently or transiently transfected or infected cells (tissue culture cells) | Vertebrate or insect cells expressing recombinant protein. |
| D2 | Insect or Xenopus cells producing recombinant protein (for example, Baculovirus vectors in insect cells or transfected/microinjected Xenopus oocytes) | |
| E1 | Yeast cells producing recombinant protein | Recombinant protein expressed in yeast cells. |
| F1 | Bacteria producing recombinant protein | Recombinant proteins expressed in bacterial cells or synthesized in vitro. |
| F2 | in vitro translation, synthetic peptides | |
| X1 | Unspecified cell type producing recombinant protein | -- |
| X2 | Unspecified material | -- |
Additional information about TRANSPATH materials, including examples, can be found here.
| Code | Method | Description/examples |
|---|---|---|
| M1 | Yeast two hybrid assay | -- |
| M2 | Screening of expression libraries with other (labeled) proteins | Phage display, eukaryotic cDNA library with EXIox vectors |
| M3 | (Co-)localization of proteins in cells and tissues by microscopical analysis | Immunofluorescence, in situ detection, electron microscopy |
| M4 | Affinity precipitation in solution/batch (with sepharose or magneto beads, for example) | Immunoprecipitation, pull-down assay |
| M5 | in vitro protein-protein binding assay (solid phase) | Enzyme Linked Immuno Sorbent Assay (ELISA) |
| M6 | Surface plasmon resonance | Detection and quantification of ligands |
| M7 | Far Western, Gel overlay, or protein-lipid overlay assay | Gel overlay: detection of binding partners on blotted proteins after transfer from a gel |
| M8 | Affinity chromatography with matrices | Proteins coupled to sepharose or agarose matrices, for example |
| M9 | Peptide spot | Search for binding partners of defined peptide motifs on a membrane |
| M10 | Cross linking | Detection by SDS-Page and Western blot |
| M11 | Electrophoretic mobility shift assay (EMSA) | -- |
| M12 | Binding assay | Binding of labeled ligand to receptor-expressing cells, quantitative evaluation, for example Scatchard analysis |
| M13 | Modification of proteins in living cellss | Protein phosphorylation and -dephosphorylation, ubiquitination, farnesylation; detection of the modified proteins from cell extracts |
| M14 | Subcellular extraction of proteins | Fractionation for localization of proteins |
| M15 | Dot spots | Spotted proteins detected with specific antibodies |
| M16 | in vitromodification of a substrate | Phosphorylation or dephosphorylation (kinase assay), ubiquitination |
| M17 | Reporter gene assay | Luciferase, beta-galactosidase assay |
| M18 | Western blot, SDS page | -- |
| M19 | mRNA detection by Northern blot | Indirect evidence for protein interaction |
| M20 | Localization of mRNA in tissues by e.g. FISH, ISH (p32, H3) | Indirect evidence for protein interaction |
| M21 | Detection of mRNA by RT-PCR | Reverse transcriptase, thereafter PCR; quantitative mRNA detection; indirect evidence for protein interaction |
| M22 | Mammalian two (or tri) -hybrid assay | -- |
| M23 | Co-sedimentation and -purification by chemico-physical methods | Biochemical purification of stable complexes by e.g analytical ultracentrifugation, size-exclusion chromatography (SEC) |
| M24 | Protein cleavage and specific degradation | Documented by detection of labeled protein fragments, immunoblots, SDS-PAGE, HPLC, mass spectral analysis |
| M25 | Interaction measurement by chemico-physical methods | Crystal structure analysis of complexes; spectrofluorometer fluorescence resonance energy transfer (FRET); intensity and/or wavelength of fluorescence of a given interacting compound changes, when another molecule binds to it; quantitative; usually done in vitro; also NMR: looking at changes in nuclear spin, usually done with peptide fragments and in unphysiological solutions, isothermal titration calorimetry (ITC) |
| M26 | mRNA expression profile analysis | Microarray data |
| M27 | Transmembrane potential measurements | Current-voltage experiments |
| M28 | Predicted by binding-site sequence | -- |
| M29 | Co-localization | -- |
| M30 | in vitro binding | -- |
| M31 | in vitro reconstitution of activity | -- |
| M32 | Flow cytometry | -- |
| M33 | Ligand binding | -- |
| M34 | Two hybrid | Cell system not specified |
| M35 | Enzyme activity measurement | -- |
| M36 | DNA footprinting/interference | -- |
| Chromatin immunoprecipitation | -- | |
| M38 | Filter binding assay | -- |
| M39 | One hybrid | Cell system not specified |
| M40 | Three hybrid | Cell system not specified |
| M41 | Osmium tetroxide modification | -- |
| M42 | Southwestern blot | -- |
| M43 | Bacterial two hybrid assay | -- |
| M44 | Bimolecular Fluorescence Complementation (BiFC) | -- |
| M45 | Immunoprecipitation of small molecule complexes with proteins e.g. receptors | -- |
| M46 | Competitive binding assay of small molecule binding | -- |
| M48 | Enzyme immunoassay (EIA) | -- |
| M49 | RNA silencing/interference | -- |
| M50 | AP-MS (affinity purification coupled with mass spectrometry) | -- |
| M51 | Bioinformatic processing | -- |
| M52 | XL-MS (chemical cross-linking coupled with mass spectrometry) | -- |
| M53 | Proximity-Labeling MS | -- |
| M54 | BioID (proximity-dependent biotin identification) | -- |
| M55 | SILAC (stable isotope labeling by/with amino acids in cell culture, based on mass spectrometry) | -- |
| M56 | AVA-Seq (all-versus-all sequencing based on a bacterial two-hybrid system) | -- |
| M57 | PCA (Protein-Fragment Complementation Assay) | -- |
Importantly, TRANSPATH not only extracts data about the materials and methods from the published literature; TRANSPATH also includes a quality evaluation and a reliability code for the material-method combinations. As such, each material and each method is given a particular symbol, and combinations of the symbols are given a reliability code, as shown in the TRANSPATH Quality Matrix.
Please note: Curators use only data from peer-reviewed publications. If controls are missing or are not credible in a specific paper, the data will not be included.