|
|
 | datum syntax: acetyl(Site(s))[DetectionMethod]
|
|
|
|
| Evidence provided: the acetylation of Subject
|
|
|
|
| Possible Subject(s): Protein, Protein Family; IP OK
|
|
|
|
| A western blot of a cell lysate, immunoprecipitate, or recombinant protein is stained with an antibody that detects a specific acetylation site when it is acetylated.
|
|
|
|
| Subject is immunoprecipitated with an antibody to an acetylation site
|
|
|
|
| A western blot of the immunoprecipitate is stained with an antibody that detects Subject
|
|
|
|
| A western blot of an anti-Subject immunoprecipitate is stained with an antibody to acetylated lysine.
|
|
|
|
| A western blot of an anti-acetylated-lysine immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| Cells are incubated in medium containing [3H]-sodium-acetate for 1 hr.
|
|
|
|
| Subject is immunoprecipitated from cell lysate and separated on an SDS-PAGE gel.
|
|
|
|
| Acetylation is determined by autoradiography.
|
|
|
|
| Cells are incubated in medium containing cycloheximide (CHX) and [3H]-sodium-acetate for 1 hr.
|
|
|
|
| Subject is immunoprecipitated from cell lysate and separated on an SDS-PAGE gel.
|
|
|
|
| Acetylation is determined by autoradiography.
|
|
|
|
| Subject is incubated with a putative acetyltransferase in the presence of 14C-acetyl-CoA.
|
|
|
|
| The amount of 32P transferred from [14C]-acetyl-CoA to Subject is detected by autoradiography.
|
|
|
|
| datum syntax: boundby[DetectionMethod]Hook
|
|
|
|
| Evidence provided: the amount of Subject bound directly by another protein(s), chemical, peptide, or nucleic acid (Hook)
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| Possible Hook(s): Protein, Family, Composite, IP, Chemical, Peptide, Oligo
|
|
|
|
| Hook is attached to a solid support.
|
|
|
|
| Subject is added and allowed to bind to Hook.
|
|
|
|
| Unbound Subject is washed away.
|
|
|
|
| The amount of bound Subject is detected by an anti-Subject antibody.
|
|
|
|
| Subject is bound to a 32P labeled oligo.
|
|
|
|
| Hook is added to Subject-oligo complex.
|
|
|
|
| Mixture is run on a Polyacrylamide gel.
|
|
|
|
| The binding of Hook to Subject is determined by the shift of Subject-oligo on the blot.
|
|
|
|
| Cell lysate or recombinant protein is separated on an SDS-PAGE gel and transferred to a WB membrane.
|
|
|
|
| Hook is added to the membrane.
|
|
|
|
| The binding of Hook to Subject is determined by the location of Subject on the blot.
|
|
|
|
| Limitation: This only measures the binding of Hook to denatured Subject.
|
|
|
|
| Subject fused to the Gal4 DNA binding domain is coexpressed with Hook fused to the the strong transcriptional activation domain of the VP16 protein and a Gal4 dependent Luciferase reporter.
|
|
|
|
| The amount of Luciferase expressed is measured by a Luciferase activity assay.
|
|
|
|
| Hook is attached to a solid support.
|
|
|
|
| Subject is added and allowed to bind to Hook.
|
|
|
|
| Unbound Subject is washed away.
|
|
|
|
| The amount of bound Subject is detected by Surface Plasmon Resonance (SPR)
|
|
|
|
| Subject and Hook are mixed together in a test tube.
|
|
|
|
| Hook is removed from the mix with anti-Hook antibodies or expression tags.
|
|
|
|
| The amount of Subject bound to Hook is determined by Western blot or autoradiography (if Subject is radiolabelled).
|
|
|
|
| Cells are incubated with labeled ligand (Hook).
|
|
|
|
| Free ligand is washed off, bound ligand is covalently cross-linked to receptors (Subjects).
|
|
|
|
| Cell Lysates are run on SDS-PAGE gels.
|
|
|
|
| Ligand bound to receptor is measured by Western blot or autoradiography (if Hook was radiolabled).
|
|
|
|
 | Chromatin Immunoprecipitation
|
|
|
|
| datum syntax: boundto(Gene)[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of Subject bound to a gene
|
|
|
|
| Possible Subject: Protein, Family, or Composite: IP not OK
|
|
|
|
| Cells are treated with formaldehyde to cross-link proteins to gene-promoters.
|
|
|
|
| Cells are lysed and chromatin is sheared by sonication or enzymic digestion.
|
|
|
|
| Subject is immunoprecipitated from sheared lysates.
|
|
|
|
| Immunoprecipitates are heated to reverse cross-links.
|
|
|
|
| Protein is removed from samples with Proteinase-K.
|
|
|
|
| Amount of Gene in the sample is determined by PCR using primers to Gene-promoter.
|
|
|
|
| datum syntax: cleavage(Site(s))[DetectionMethod]
|
|
|
|
| Evidence provided: the cleavage of Subject
|
|
|
|
| Possible Subject(s): Protein or Family; IP OK
|
|
|
|
| A western blot of a cell lysate, immunoprecipitate, or recombinant protein is stained with an antibody that recognizes the cleavage product of subject.
|
|
|
|
| A western blot of a cell lysate, immunoprecipitate, or recombinant protein is stained with an antibody that recognizes the cleavage product of subject cleaved at a specific site.
|
|
|
|
| datum syntax: colocwith[DetectionMethod]Hook
|
|
|
|
| Evidence provided: the colocalization of Subject with Hook
|
|
|
|
| Possible Subject(s): Proteins, Family, or Composites; IP not OK
|
|
|
|
| Cells are transfected with plasmids expressing Subject and/or Hook fused to fluorescent proteins or stained with fluorescent antibodies against Subject and/or Hook.
|
|
|
|
| Images of staining patterns of Subject and Hook are superimposed.
|
|
|
|
| A change in the color of both markers indicates colocalization.
|
|
|
|
| Cells are transfected with plasmids expressing Subject fused to a donor fluorophore and Hook fused to an acceptor fluorophore or stained with antibodies labeled with donor and acceptor fluorophores against Subject and Hook.
|
|
|
|
| Colocalization (energy transfer) is detected as an increase in donor fluorescence (dequenching) after complete photobleaching of the acceptor molecule.
|
|
|
|
| Cells are stained with antibodies against Subject and Hook.
|
|
|
|
| Images are made using an electron microscope.
|
|
|
|
| Colocalization is determined as defined by observer.
|
|
|
|
| datum syntax: copptby[DetectionMethod]Hook
|
|
|
|
| Evidence provided: the amount of Subject coprecipitated by Hook
|
|
|
|
| Possible Subject(s): Proteins, Family, or Composites; IP not OK
|
|
|
|
| Hook is immunoprecipitated from a cell lysate.
|
|
|
|
| A western blot of the immunoprecipitate is stained for Subject.
|
|
|
|
| Cells are treated with a chemical crosslinker before lysis.
|
|
|
|
| Hook is immunoprecipitated from cell lysate.
|
|
|
|
| A western blot of the immunoprecipitate is stained for Subject.
|
|
|
|
| datum syntax: Gal4-reporter[DetectionMethod]
|
|
|
|
| Evidence provided: the activity of a transcripton factor (Subject)
|
|
|
|
| Possible Subject(s): expressed Protein fused to Gal4-DNA-binding domain
|
|
|
|
| Cells are transfected with a plasmid expressing the Subject fused to the DNA binding domain of Gal4 and another plasmid expressing Luc or CAT driven by a promoter containing a Gal4 response element.
|
|
|
|
| The amount of [Luc] or [CAT] expressed is measured by activity assays.
|
|
|
|
| datum syntax: GDP-dissociation[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of GDP released from Subject
|
|
|
|
| Possible Subject(s): recombinant Protein
|
|
|
|
| Subject is preloaded with labeled GDP.
|
|
|
|
| Subject is incubated with a putative guanine nucleotide exchange factor.
|
|
|
|
| The amount of GDP released is detected by:
|
|
|
|
| liquid scintillation counting [3H-GDP]
|
|
|
|
| datum syntax: GTP-association[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of GTP bound to Subject
|
|
|
|
| Possible Subject(s): recombinant Protein or IP
|
|
|
|
| Using [35S-GTPgS] [32P-GTP] or [Mant-GTP]
|
|
|
|
| Subject is incubated with labeled GTP and a putative guanine nucleotide exchange factor.
|
|
|
|
| Subject is bound to nitrocellulose and washed to removed unbound GTP
|
|
|
|
| The amount of GTP bound is detected by:
|
|
|
|
| liquid scintillation counting (for 35S-GTP or 32P-GTP)
|
|
|
|
| Cherenkov counting (for 32P-GTP)
|
|
|
|
| fluorimeter (for Mant-GTP)
|
|
|
|
| The GTPase binding domain of a GTPase effector is added to a cell lysate.
|
|
|
|
| The GTPase binding domain is precipitated from a cell lysate.
|
|
|
|
| A western blot of the immunoprecipitate is stained for Subject.
|
|
|
|
| Cells are transfected with plasmids expressing a chimeric protein consisting of a GTP-binding protein (Subject), the binding domain of a protein that only binds to Subject bound to GTP, and yellow-emitting (YFP) and cyan-emitting (CFP) GFP mutants.
|
|
|
|
| GTP binding to Subject increases the efficiency of FRET between CFP and YFP.
|
|
|
|
| Cells are preincubated with radioactive inorganic phosphate (32Pi).
|
|
|
|
| Subject is immunoprecipitated from cell lysates.
|
|
|
|
| Nucleotides are eluted from the immunoprecipitates and separated by TLC.
|
|
|
|
| The amount of radioactivity in the GTP vs GDP spots is calculated.
|
|
|
|
| datum syntax: GTP-hydrolysis[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of GTP hydrolyzed by Subject
|
|
|
|
| Possible Subject(s): recombinant Protein
|
|
|
|
| Subject is preloaded with 32P-GTP.
|
|
|
|
| Subject is incubated with a putative GTPase activating factor.
|
|
|
|
| Subject is bound to nitrocellulose and washed to removed unbound GTP
|
|
|
|
| The amount of 32P released (total 32P added - bound 32P) is determined by liquid scintillation or Cherenkov counting.
|
|
|
|
| Subject is preloaded with GTP.
|
|
|
|
| Subject is incubated with a putative GTPase activating factor.
|
|
|
|
| The amount of Pi released is determined by the MESG/phosphorylase system [9268338] which monitors free gamma-Pi release from bound GTP.
|
|
|
|
| Subject is preloaded with 32P-GTP.
|
|
|
|
| Subject is incubated with a putative GTPase activating factor.
|
|
|
|
| Bound nucleotides are separated by TLC.
|
|
|
|
| Total bound 32P is determined by liquid scintillation or Cherenkov counting.
|
|
|
|
| Percent GTP is calculated.
|
|
|
|
| datum syntax: infraction(Fraction)[DetectionMethod]
|
|
|
|
| Evidence provided: the existence of the Subject in a fraction of the cell lysate
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| cytoplasmic cytosolic cytoskeleton membrane nuclear P100 PM2 S100 DRM cyto/nuc
|
|
|
|
| Cells are lysed and fractionated
|
|
|
|
| The amount of Subject in each fraction is determined by Western blot.
|
|
|
|
| datum syntax: internalization[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of Subject internalized (no longer expressed on the cell surface)
|
|
|
|
| Possible Subject(s): Protein, Family, Composite, or Chemical; IP not OK
|
|
|
|
| Cells are stained with antibodies to Subject.
|
|
|
|
| Location of Subject is determined by microcopy.
|
|
|
|
| A change from cell-membrane staining to cytoplasmic staining is an indication of internalization of Subject.
|
|
|
|
| Subject ligand is covalently labelled with 125I or another marker such as biotin
|
|
|
|
| Labeled ligand is added to cells causing the receptor to internalize with the ligand.
|
|
|
|
| Ligand remaining on the cell surface is removed by washing with high salt and/or low pH.
|
|
|
|
| Remaining radioactivity is counted in a gamma-counter.
|
|
|
|
| datum syntax: IVGefA(Substrate(s))[DetectionAssay]
|
|
|
|
| Evidence provided: the ability of the complex immunoprecipitated with Subject to cause exchange of GDP to GTP from a substrate
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP required
|
|
|
|
| Possible Substrate(s): Protein
|
|
|
|
| An immunoprecipitate is incubated with a recombinant GTP-binding Protein preloaded with labeled GDP
|
|
|
|
| The amount of GDP released is detected by:
|
|
|
|
| liquid scintillation counting [3H-GDP]
|
|
|
|
| fluorimeter [for Mant-GDP]
|
|
|
|
| datum syntax: IVHatAct[DetectionAssay]
|
|
|
|
| Evidence provided: the ability of the complex immunoprecipitated with Subject to acetylate histones
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP required
|
|
|
|
| An immunoprecipitate is incubated with histones in the presence of 14C-acetyl-CoA.
|
|
|
|
| The amount of 32P transferred from [14C-acetyl-CoA] to histones is detected by autoradiography.
|
|
|
|
| An immunoprecipitate is incubated with biotinylated histone H4 peptide in the presence of acetyl-CoA.
|
|
|
|
| An aliquot of the reaction is immobilized onto streptavidin plates and histone acetylation detected using a ELISA.
|
|
|
|
| datum syntax: IVKA(Substrate(s))(Site(s))[DetectionMethod] for Protein kinases
|
|
|
|
| datum syntax: IVLKA(Substrate(s))[DetectionMethod] for Lipid kinases
|
|
|
|
| Evidence provided: the ability of the complex immunoprecipitated with Subject to phosphorylate a substrate
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP or recombinant Protein required
|
|
|
|
| Possible Substrate(s): Protein(s), Peptide, auto (for Protein kinases), lipid (for Lipid Kinases)
|
|
|
|
| More than one Protein (separated by commas) can be used as substrate to represent coupled reactions.
|
|
|
|
| Site(s) are only used when phosAb is used as a detection method.
|
|
|
|
| An immunoprecipitate or recombinant protein is incubated with substrate in a kinase buffer containing required co-factors and 32P-ATP.
|
|
|
|
| The reaction mixture is separated on a SDS-PAGE gel. The amount of 32P transferred from 32P-ATP to the substrate is detected by autoradiography.
|
|
|
|
| An immunoprecipitate or recombinant protein is incubated with substrate in a kinase buffer containing required co-factors and ATP.
|
|
|
|
| The reaction mixture is separated on a SDS-PAGE gel.
|
|
|
|
| The phosphorylation state of the substrate is determined using an antibody that detects a specific phosphorylation site when it is phosphorylated.
|
|
|
|
| An immunoprecipitate or recombinant protein is incubated with substrate in a kinase buffer containing required co-factors and ATP.
|
|
|
|
| The reaction mixture is separated on a SDS-PAGE gel.
|
|
|
|
| The phosphorylation state of the substrate is determined using an antibody to phosphotyrosine.
|
|
|
|
| An immunoprecipitate or recombinant protein is separated on a SDS-PAGE gel.
|
|
|
|
| The gel is incubated with substrate in a kinase buffer containing required co-factors and 32P-ATP.
|
|
|
|
| The phosphorylation state of the substrate is determined by autoradiography.
|
|
|
|
| An immunoprecipitate is incubated with lipid-substrate in a kinase buffer containing required co-factors and 32P-ATP.
|
|
|
|
| Reaction components are separated by Thin Layer Chromatography (TLC).
|
|
|
|
| Production of phosphorylated lipid substrate is measured by autoradiography.
|
|
|
|
| In Vitro Phosphatase Activity
|
|
|
|
| datum syntax: IVPPaseAct(Substrate(s))(Site(s))[DetectionMethod]
|
|
|
|
| Evidence provided: the ability of the complex immunoprecipitated with Subject to phosphorylate a substrate
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP or recombinant Protein required
|
|
|
|
| An immunoprecipitate or recombinant protein is incubated with para-Nitrophenylphosphate in a phosphatase buffer containing required co-factors and inhibitors.
|
|
|
|
| Reaction (production of para-nitrophenol (yellow) is monitored by absorbance at 405 nm.
|
|
|
|
| An immunoprecipitate or recombinant protein is incubated with substrate in a phosphatase buffer containing required co-factors and inhibitors.
|
|
|
|
| The reaction mixture is separated on a SDS-PAGE gel.
|
|
|
|
| The phosphorylation state of the substrate is determined using an antibody that detects a specific phosphorylation site when it is phosphorylated.
|
|
|
|
| datum syntax: locatedin(Position)[DetectionMethod]
|
|
|
|
| Evidence provided: the location of the Subject in a cell
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP not OK
|
|
|
|
| Possible Positions: cell-membrane, nucleus, cytoplasm, perinuclear-region, subnuclear-bodies, DSBs
|
|
|
|
| Cells are stained for Subject.
|
|
|
|
| Position of Subject in respect to cell is determine by microscopy
|
|
|
|
| datum syntax: methyl(Site(s))[DetectionMethod]
|
|
|
|
| Evidence provided: the methylation of Subject
|
|
|
|
| Possible Subject(s): Protein, Protein Family; IP OK
|
|
|
|
| Possible DetectionMethod(s): 3H-SAM MeAb MeAb-ppt RMeAb RMeAb-ppt
|
|
|
|
| Subject is incubated with 3H-labeled S-adenosyl methionine and a histone methyltransferase.
|
|
|
|
| The reaction mixture is separated on a SDS-PAGE gel.
|
|
|
|
| The amount of 3H transferred from 3H-SAM to Subject is detected by autoradiography.
|
|
|
|
| A western blot of a cell lysate or immunoprecipitate is stained with an antibody that detects a specific methylation site when it is methylated.
|
|
|
|
| Subject is immunoprecipitated with an antibody to a methylation site
|
|
|
|
| A western blot of the immunoprecipitate is stained with an antibody that detects Subject
|
|
|
|
| A western blot of a cell lysate or immunoprecipitate is stained with an antibody that detects mono/di-methylarginine.
|
|
|
|
| Subject is immunoprecipitated with an antibody to mono/di-methylarginine.
|
|
|
|
| A western blot of the immunoprecipitate is stained with an antibody that detects Subject.
|
|
|
|
| datum syntax: mRNA[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of mRNA expressed by Subject
|
|
|
|
| Possible Subject(s): Gene
|
|
|
|
| Total RNA is prepared from the cells.
|
|
|
|
| Relative amount of mRNA is detected using a Northern Blot.
|
|
|
|
| Total RNA is prepared from the cells.
|
|
|
|
| Relative amount of mRNA is detected using a RNAse Protection Assay.
|
|
|
|
| Total RNA is prepared from the cells.
|
|
|
|
| Relative amount of mRNA is detected using Reverse Transcription Polymerase Chain Reaction.
|
|
|
|
| Total RNA is prepared from the cells.
|
|
|
|
| Relative amount of mRNA is detected using a DNA Microarray.
|
|
|
|
| datum syntax: nedd[DetectionMethod]
|
|
|
|
| Evidence provided: the neddylation of Subject
|
|
|
|
| Possible Subject(s): Protein, Protein Family; IP OK
|
|
|
|
| A western blot of an anti-Subject immunoprecipitate is stained with an antibody to Nedd8 (NdAb), or a tag on expressed or recombinant Nedd8 (tNdAb) .
|
|
|
|
| Using [NdAb-ppt] or [tNdAb-ppt]:
|
|
|
|
| A western blot of an anti-Nedd8 or anti-Nedd8-tag immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| A western blot of a cell lysate or immunoprecipitate is stained with an antibody to Subject. A reduction in Subject mobility is indicative of neddylation.
|
|
|
|
| datum syntax: nuc-export[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of Subject exported from the nucleus
|
|
|
|
| Possible Subject(s): BProtein or Family; IP not OK
|
|
|
|
| Subject is imported into digitonin permeabilized cells (see nuclear import using IHC)
|
|
|
|
| Subject is allowed to export nuclei in the presence of transport buffer
|
|
|
|
| Sometimes cytosols and/or energy mixes are added to transport buffer
|
|
|
|
| Cells are transfected with a plasmid expressing the Subject to Enterobacteria phage MS2 coat protein and a reporter plasmid encoding CAT or Luc and the bacteriophage MS2 translational operator RNA, with these two elements flanked by a pair of splice acceptor and donor sites.
|
|
|
|
| Expression of CAT or Luc requires active nuclear export of the unspliced CAT or Luc mRNA.
|
|
|
|
| The amount of CAT or Luc expressed is measured by activity assays.
|
|
|
|
| Using [HeterokaryonAssay]:
|
|
|
|
| GSN2 cells expressing Subject are fused with NIH3T3 cells using polyethylene glycol in the presence of CHX to block protein synthesis.
|
|
|
|
| Cells are stained with Hoechst dye 33258 which distinguishes GSN2 nuclei (smooth nuclear staining) from NIH3T3 nuclei (punctate nuclear staining).
|
|
|
|
| Cells are examined by fluorescence microscopy.
|
|
|
|
| The presence of Subject in NIH3T3 nuclei is indicative of nuclear export.
|
|
|
|
| datum syntax: nuc-import[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of Subject imported into the nucleus
|
|
|
|
| Possible Subject(s): recombinant Protein
|
|
|
|
| Adherent cells are permeabilized with digitonin and washed to remove cytoplasmic proteins.
|
|
|
|
| Purified Subject is added to permeabilized cells.
|
|
|
|
| Sometime "import mix" (ATP, GTP, creatine phosphate, and creatine phosphokinase) is added with subject to provide the energy required for import
|
|
|
|
| Sometime soluble transport factors are added.
|
|
|
|
| After import reaction, the cells were washed and immunostained for Subject.
|
|
|
|
| Subject is mixed with cytosolic extracts to provide Kpnas and Kpnb1 and added to cells treated with digitonin to permeabilize the plasma membrane but leave nuclear membranes intact.
|
|
|
|
| The amount of Subject imported into the nucleus is determined by immunohistochemistry or Western blot of nuclear fractions.
|
|
|
|
| datum syntax: oligo-binding[DetectionMethod]
|
|
|
|
| Evidence provided: the ability of the Subject to bind a DNA oligo containing at least one copy of its consensus binding sequence.
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP not OK
|
|
|
|
| the name and sequence of the oligo are supplied in the oligo field if available
|
|
|
|
| Nuclear extract, recombinant protein, or IP is incubated with a radiolabeled oligonucleotide and run on a Polyacrylamide gel.
|
|
|
|
| Migration of the oligonucleotide is determined by autoradiograpy.
|
|
|
|
| Binding of protein to the oligonucleotide is indicated by a changed in the mobility of the oligonucleotides.
|
|
|
|
| The identity of Subject is inferred by the sequence of the oligonucleotide or by supershift after addition of anti-Subject antibody to protein:oligo complex.
|
|
|
|
| Nuclear extracts or total lysates are incubated with immobilized oligonucleotide.
|
|
|
|
| Non-binding proteins are washed off and remaining proteins are analyzed by ELISA.
|
|
|
|
| datum syntax: oligomerization[DetectionMethod]
|
|
|
|
| Evidence provided: the oligomerization of Subject
|
|
|
|
| Possible Subject(s): Protein or Family; IP OK
|
|
|
|
| Subject is expressed in cells using two different expression tags.
|
|
|
|
| Subject is immunopreciptiated using one expression tag.
|
|
|
|
| A western blot of the immunoprecipitate is stained with an antibody to the second expression tag.
|
|
|
|
| Using [density-gradient]:
|
|
|
|
| Subject (recombinant protein only) is fractionated by density-gradient ultracentrifugation.
|
|
|
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| Fractions are collected, run on SDS-PAGE gels, blotted, and stained for Subject
|
|
|
|
| Subject fused to a fluorescent protein is expressed in cells.
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| Cells are stained with a fluorescent antibody to Subject.
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| Images of staining pattern of Subject-fluorescent-protein and Subject-fluorescent-antibody are superimposed.
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|
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| A change in the color of both markers indicates oligomerization.
|
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|
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| Lysates or immunoprecipitates are run on non-denaturing PAGE gels, blotted, and stained for Subject.
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|
| Limitations: Does not distinquish between a complexes of Subject:Subject and Subject:OtherProtein of the same size.
|
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|
|
| Cells are metabolically labelled with 35S methionine/cyteine
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|
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| Cells are stimulated followed by treatment with a crosslinking agent
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|
|
| Cells are lysed, run on SDS-PAGE gels, blotted and stained for Subject
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|
|
| datum syntax: phos(Site(s))[DetectionMethod]
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|
| Evidence provided: the phosphorylation of Subject
|
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| Possible Subject(s): Protein or Family; IP OK
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|
|
| Possible DetectionMethod(s): 2D-PPMap 32P-ATP 32Pi WBMS WBMS/PPase phosAb phosAb-ppt HPLC-PAA-ED
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|
|
| Subject is phosphorylatated in an In Vitro Kinase assay using 32P-ATP.
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|
|
|
| Phosphorylation site(s) is determined by two-dimensional phosphopeptide mapping.
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|
|
| InCell phosphosphorylation
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|
|
| Cells are preincubated with radioactive inorganic phosphate (32Pi).
|
|
|
|
| Subject is immunoprecipitated and analyzed for site phosphorylation by two-dimensional phosphopeptide mapping
|
|
|
|
| Subject is incubated with a putative kinase (Treatment) in a kinase buffer containing required co-factors and 32P-ATP.
|
|
|
|
| The reaction mixture is separated on a SDS-PAGE gel.
|
|
|
|
| The amount of 32P transferred from 32P-ATP to Subject is detected by autoradiography.
|
|
|
|
| Cells are preincubated with radioactive inorganic phosphate (32Pi).
|
|
|
|
| Subject is immunoprecipitated and run on an SDS-PAGE gel.
|
|
|
|
| The amount of 32P in the immunoprecipitate is detected by autoradiography.
|
|
|
|
| Cells are preincubated with radioactive inorganic phosphate (32Pi).
|
|
|
|
| Subject is immunoprecipitated and digested with trypsin.
|
|
|
|
| Tryptic phosphopeptides are analyzed for site phosphorylation by HPLC followed by phosphoamino acid analysis and Edman degradation.
|
|
|
|
| Subject is immunoprecipitated with an antibody to a phosphorylation site
|
|
|
|
| A western blot of the immunoprecipitate is stained with an antibody that detects Subject
|
|
|
|
| A western blot of a cell lysate or immunoprecipitate is stained with an antibody that detects a specific phosphorylation site when it is phosphorylated.
|
|
|
|
| A western blot of a cell lysate or immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| Phosphorylation is implied by a decrease in the mobility shift of Subject.
|
|
|
|
| A western blot of a cell lysate or immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| Phosphorylation is implied by a decrease in the mobility shift of Subject.
|
|
|
|
| Phosphorylation is confirmed by a loss of mobility shift in response to treating lysate or immunoprecipitate with a phosphatase before western blot.
|
|
|
|
| datum syntax: Sphos[DetectionMethod]
|
|
|
|
| Evidence provided: the phosphorylation of Subject on serine
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| A western blot of an anti-Subject immunoprecipitate is stained with an antibody to phosphoserine.
|
|
|
|
| A western blot of an anti-phosphoserine immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| Cells are preincubated with radioactive inorganic phosphate (32Pi).
|
|
|
|
| Subject is immunoprecipitated and run on an SDS-PAGE gel.
|
|
|
|
| The band containing Subject is cut out of the gel and analyzed for serine phosphorylation by Phosphoamino Acid Analysis.
|
|
|
|
| datum syntax: STphos[DetectionMethod]
|
|
|
|
| Evidence provided: the phosphorylation of Subject on serine and threonine
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| A western blot of an anti-Subject immunoprecipitate is stained with an antibody to phosphoserine/threonine.
|
|
|
|
| A western blot of an anti-phosphoserine/threonine immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| Cells are preincubated with radioactive inorganic phosphate (32Pi).
|
|
|
|
| Subject is immunoprecipitated and run on an SDS-PAGE gel.
|
|
|
|
| The band containing Subject is cut out of the gel and analyzed for serine and threonin phosphorylation by Phosphoamino Acid Analysis.
|
|
|
|
| datum syntax: Tphos[DetectionMethod]
|
|
|
|
| Evidence provided: the phosphorylation of Subject on threonine
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| A western blot of an anti-Subject immunoprecipitate is stained with an antibody to phosphothreonine.
|
|
|
|
| A western blot of an anti-phosphothreonine immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| Cells are preincubated with radioactive inorganic phosphate (32Pi).
|
|
|
|
| Subject is immunoprecipitated and run on an SDS-PAGE gel.
|
|
|
|
| The band containing Subject is cut out of the gel and analyzed for serine and threonin phosphorylation by Phosphoamino Acid Analysis.
|
|
|
|
| datum syntax: Yphos[DetectionMethod]
|
|
|
|
| Evidence provided: the phosphorylation of Subject on tyrosine
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| A western blot of an anti-Subject immunoprecipitate is stained with an antibody to phosphotyrosine.
|
|
|
|
| A western blot of an anti-phosphotyrosine immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| Cells are preincubated with radioactive inorganic phosphate (32Pi).
|
|
|
|
| Subject is immunoprecipitated and run on an SDS-PAGE gel.
|
|
|
|
| The band containing Subject is cut out of the gel and analyzed for serine and threonin phosphorylation by Phosphoamino Acid Analysis.
|
|
|
|
| datum syntax: polymerization[DetectionMethod]
|
|
|
|
| Evidence provided: the polymerization of Subject
|
|
|
|
| Possible Subject(s): Protein or Family; IP OK
|
|
|
|
| Subject is detected by Western Blot [WB].
|
|
|
|
| A long smear rather than a single band is indicative of polymerization
|
|
|
|
| datum syntax: promo-reporter[DetectionMethod]
|
|
|
|
| Evidence provided: the promoter activity of the Subject
|
|
|
|
| Possible Subject(s): Gene
|
|
|
|
| Cells are transfected with a plasmid expressing [Luc] or [CAT] driven by the promoter of the Subject.
|
|
|
|
| The amount of Luc or CAT expressed is measured by activity assays.
|
|
|
|
| datum syntax: prot-exp[DetectionMethod]
|
|
|
|
| Evidence provided: the relative amount of Subject expressed
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| A western blot is stained with an antibody to Subject.
|
|
|
|
| Cells are permeabilized, stained with an antibody to Subject, and analyzed by FACS.
|
|
|
|
| Cells are preincubated with 35S-methionine and/or cysteine
|
|
|
|
| Subject is immunoprecipitated and run on an SDS-PAGE gel, and detected by autoradiography.
|
|
|
|
| Cells lysates are incubated with an immobilized antibody to Subject.
|
|
|
|
| The amount of Subject bound to immobilized antibody is determine by staining with another antibody to Subject.
|
|
|
|
| datum syntax: prot-stability[DetectionMethod]
|
|
|
|
| Evidence provided: the stabilty of the Subject
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| Cellular proteins are metabolically labeled with 35S.
|
|
|
|
| 35S is replaced with unlabelled amino acids and incubated for various times.
|
|
|
|
| Subject is immunoprecipitated and run on an SDS-PAGE gel, and detected by autoradiography.
|
|
|
|
| Protein stability is calculated from the decrease in signal over time.
|
|
|
|
| Cells are treated with cycloheximide (CHX) to prevent protein synthesis.
|
|
|
|
| Amount of Subject remaining over time is monitored by Western blot.
|
|
|
|
| datum syntax: secretion[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of Subject secreted by cells
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| The concentration of Subject in the supernatant is analyzed by:
|
|
|
|
| Enzyme-linked immunosorbent assay [ELISA]
|
|
|
|
| an ELISA performed on suspended bead and analysed by FACS [BeadArray]
|
|
|
|
| datum syntax: snaggedby[DetectionMethod] Hook
|
|
|
|
| Evidence provided: the amount of Subject "pulled-down" from a lysate by Hook
|
|
|
|
| Possible Subject: Protein, Family, or Composites; IP not OK
|
|
|
|
| Possible Hook: recombinant or purified Protein, Chemical, Oligo
|
|
|
|
| Hook is added to a cell lysate.
|
|
|
|
| Hook is precipitated from a cell lysate.
|
|
|
|
| A western blot of the precipitate is stained for Subject.
|
|
|
|
| datum syntax: sumo[DetectionMethod]
|
|
|
|
| Evidence provided: the sumoylation of Subject
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| A western blot of an anti-Subject immunoprecipitate is stained with an antibody to Sumo (SuAb) or a tag on expressed Sumo (tSuAb).
|
|
|
|
| Using [SuAb-ppt] or [tSuAb-ppt]:
|
|
|
|
| A western blot of an anti-Sumo or anti-Sumo-tag immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| A western blot of a cell lysate or immunoprecipitate is stained with an antibody to Subject. A reduction in Subject mobility is indicative of sumoylation.
|
|
|
|
| datum syntax: surface-exp[DetectionMethod]
|
|
|
|
| Evidence provided: the amount of Subject located on the surface of a cell
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP not OK
|
|
|
|
| The concentration of Subject on the surface of a cell is analyzed by FACS.
|
|
|
|
| A ligand specific for Subject is added to cells on ice.
|
|
|
|
| The amount of bound ligand is determined by scintillation counting.
|
|
|
|
| All the proteins on the surface of the cell are cross-linked to NHS-SS-biotin.
|
|
|
|
| Cells are washed to remove unreacted reagent, lysed, and precipitated with antibody against Subject.
|
|
|
|
| Precipitated proteins are analysed by western blot and detected using labeled avidin.
|
|
|
|
| datum syntax: ubiq[DetectionMethod]
|
|
|
|
| Evidence provided: the ubiquitination of Subject.
|
|
|
|
| Detection with K63UbAb is provides addition evidence that the bound ubiquitin is K63 linked.
|
|
|
|
| Possible Subject(s): Protein, Family, or Composite; IP OK
|
|
|
|
| Using [UbAb], [tUbAb], or [K63UbAb]:
|
|
|
|
| A western blot of an anti-Subject immunoprecipitate is stained with an antibody to ubiquitin, K63-linked polyubiquitin, or a tag on expressed or recombinant ubiquitin .
|
|
|
|
| Direct ubiquitination of subject can be determined by boiling lysates before immunoprecipitation to remove noncovalently attached proteins.
|
|
|
|
| Using [UbAb-ppt] or [tUbAb-ppt]:
|
|
|
|
| A western blot of an anti-Ubiquitin or anti-Ubiquitin-tag immunoprecipitate is stained with an antibody to Subject.
|
|
|
|
| Direct ubiquitination of subject can be determined by boiling lysates before immunoprecipitation to remove noncovalently attached proteins.
|
|
|
|
| A western blot of a cell lysate or immunoprecipitate is stained with an antibody to Subject. A reduction in Subject mobility is indicative of ubiquitination.
|
|
|
|
| datum syntax: upshift[DetectionMethod]
|
|
|
|
| Evidence provided: Subject is modified in some way that causes a decrease in its mobility on a Western blot.
|
|
|
|
| Possible Subject(s): Protein or Family; IP OK
|
|
|
|
| A western blot of a cell lysate, immunoprecipitate, or recombinant protein is stained with an antibody that recognizes subject.
|
|
|
|
| A reduction in Subject mobility is indicative of some sort of covalent modification.
|
|
|
|
|
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