Affinity chromatography based strategies “Fishing for partners” Immunoprecipitation approaches The TAP TAG system Two hybrid assay.

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Presentation transcript:

Affinity chromatography based strategies “Fishing for partners” Immunoprecipitation approaches The TAP TAG system Two hybrid assay

Yeast Gal4 transcription factor Zinc Finger DNA binding domain (DBD) Transcriptional Activation Domain (AD) AD DBD Gene X ON promoter Yeast Two Hybrid Assay

Immuno Precipitation & Pull- Down Experiments bait protein complexed proteins IgG Band Highly specific antibody recognizing bait protein required! Use of recombinant bait protein: pull-down experiment

eIF4E complex in Drosophila The eukaryotic translation initiation factor 4E (eIF4E) directly binds the 7-methyl-guanosine cap structure at the 5’ end of all eukaryotic mRNAs. eIF4E recruits additional factors to form a complex that, after mRNA circularization, initiates translation m7G eIF4G AUG Stimulating factors eIF4E AAAAAAA PAB CUP

LCMSMS identification of Immunoprecipitated molecular partners of eIF4E GFEDCBAGFEDCBA IP -  eIF4E Marker Banda Proteins Hsp-83 Hsc70-4 EF-2 eEF-1 -  eEF-1 -  Cup eIF4G eIF4A CG PA PABP Molecular Partners of eIF4E already known New molecular partners of eIF4E D B G C F B E C E

Tandem affinity purification (TAP) strategy

Figure 3 Primary validation of complex composition by 'reverse' purification: the polyadenylation machinery. a, A similar band pattern is observed when different components of the polyadenylation machinery complex are used as entry points for affinity purification. Underlined are new components of the polyadenylation machinery complex for which a physical association has not yet been described. The bands of the tagged proteins are indicated by arrowheads. b, Proposed model of the polyadenylation machinery.

Affinity Tags and Ligands Protein System Ligand Histidine Tail Ni 2+ Biotinyl Tag Streptavidin GST-fused Glutathione Specific Epitope Monoclonal Ab

Protein Bait Specific ligand on beads Bait linked to the beads Specific Interactions Cellular extract incubated with the immobilised bait Unbound proteins Elution of bait and partner(s) SDS-PAGE separation Fishing for Partners Strategy MS Identification

Disruption of intracellular compartments Altered bait/partners molar ratios Essentially in vitro interactions Pre-cleaning of the samples required Very good control needed False Positives

Interaction Proteomics by Immunoprecipitation Methods plasmid expressing epitope- tagged bait protein bait Immuno precipitate protein complex Fractionate on 1-D gel Identify components by mass spec Transfect into cells

Immunoprecipitation of FLAG-FCP1 FLAGFCP1 FLAG FCP1 Y Z W Anti-FLAG Agarose bead SDS-PAGE separation MS Identification

Fishing for DNA-binding protein MS Identification Multimers of the target oligonucleotide linked to a support Unbound proteins Specific DNA-protein(s) Interactions Nuclear extract incubated in batch with the immobilised bait SDS-PAGE separation Elution of bound protein(s)

Identification of the putative transcriptional repressor 80 kDa In situ digestion T° gi| |ref|NP e zinc finger protein ® [Homo sapiens] e T° gi| |ref|XP minichromosome maintenance ® deficient (mis5, S. pombe) 6 [Homo sapiens]