BIOC23H3 Lecture : Lec 12 - Analysis of Protein-Protein Interactions.doc
2 May 2012
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Methods for protein-protein interaction analysis: 1) size exclusion chromatography, 2) ap-ms, affinity purification coupled w/ mass spec, tandem affinity purification (tap) tag used. Hsp90 bound to tap-tap (protein a and calmodulin binding peptide) The lysate is put through igg resin where protein a will bind the resin. After cleavage, you have hsp90 bound to only calmodulin binding peptide; put this through resin of calmodulin beads. Now, you"ve purified the hsp90-calmodulin binding peptide protein along with hsp90"s interactors. Run sds-page: will get hsp90 band and several other protein bands of diff sizes, can analyze these diff proteins using mass spec. Trypsin digest and then tandem mass spec. Transcription factor = 2 domains: dna binding domain (dbd) = dna binding, transactivation domain (ad) = transcription. Dbd and ad = separate and fused to 2 diff interest proteins respectively and expressed in the 2 diff mating type yeast cells.
