2013 分子生物工作基础 CLASS 1.pptVIP

Two-hybrid screens are low-tech; they can be carried out in any lab without sophisticated equipment. Two-hybrid screens can provide an important first hint for the identification of interaction partners. The assay is scalable, which makes it possible to screen for interactions among many proteins. Yeast two-hybrid data can be of similar quality to data generated by the alternative approach of coaffinity purification followed by mass spectrometry (AP/MS). Y2H Strengths Y2H Weaknesses A high number of false positive (and false negative) identifications. The hybrid proteins are fusion proteins, and the fused parts may inhibit certain interactions. An interaction may not happen in yeast, the typical host organism for Y2H. The Y2H takes place in the nucleus. If test proteins are not localized to the nucleus, two interacting proteins may be found to be non-interacting. Some proteins might specifically interact when they are co-expressed in the yeast, although in reality they are never present in the same cell at the same time. Phage Display Phage vector DNA LIGATION DNA encoding peptide of interest INFECT E. coli fusion library of many phage, each with a different peptide displayed on its surface Gene encoding a phage coat protein Peptide in fusion protein displayed on surface of phage AFFINITY CHROMATOGRAPHY ELUTE CHARACTERIZATION Proteins to Genes Protein-protein interaction Proteomics Affinity purification GST PULL-DOWN (In vitro) Y Z Cell extracts Sepharose 4B GST BAIT Glutathione Sepharose 4B GST BAIT x X Y Z SDSMass spectrometry Protein sequence Nucleotide sequence Christopher Rachez et al. 1999 Nature 398, 824 - 828 FLAG System Affinity Purification (In vivo) Transfect cells with plasmids encoding FLAG-tagged protein of interest Collect cell lysates with IP buffer containing 0.1%-0.5% NP-40 Affinity purification using ANTI-FLAG? M2 Affinity Gel SDS, silver staining, cut bands of interest, determine t