Download Applications of Chimeric Genes and Hybrid Proteins - Part C: by Melvin I. Simon, Jeremy Thorner, Scott D. Emr, John N. PDF

By Melvin I. Simon, Jeremy Thorner, Scott D. Emr, John N. Abelson

The seriously acclaimed laboratory typical for greater than 40 years, Methods in Enzymology is likely one of the so much hugely revered guides within the box of biochemistry. considering 1955, every one quantity has been eagerly awaited, usually consulted, and praised via researchers and reviewers alike. Now with greater than three hundred volumes (all of them nonetheless in print), the sequence comprises a lot fabric nonetheless suitable today--truly an important ebook for researchers in all fields of existence sciences

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Extra resources for Applications of Chimeric Genes and Hybrid Proteins - Part C: Protein-Protein Interactions and Genomics

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Systems FINLEY, JR. Introduction The yeast two-hybrid system is a powerful assay for protein-protein interactions. 1 As described in chapters 1, 2, 6, and 10 of this volume, several versions of the two-hybrid system have been developed. Most versions have the following features. The two proteins to be tested for interaction are expressed as hybrids in the nucleus of a yeast cell. One of the proteins is fused to the DNA-binding domain (DBD) of a transcription factor and the other is fused to a transcription activation domain (AD).

Winston, and P. " Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1990. 10 TWO-HYBRID METHODS AND RELATED TECHNIQUES [1] pendent colonies per transformation for each array element. The pooling of too many colonies risks the possibility of variants outcompeting the desired one in culture. We determined experimentally that the sum of all errors introduced in the cloning process was approximately 10% (oligonucleotide synthesis, PCR, recombination cloning) and reasoned that the presence of two independent colonies would increase the probability that at least one correctly synthesized fusion protein would be present per array element.

The truncated fusion proteins will, it is hoped, retain many binding characteristics of the full-length protein, but lose the high transcriptional activity. The truncated fusion protein approach is particularly useful if Bait alone vigorously activates transcription. LexA system II also provides a potentially significant advantage in that inclusion of 3-AT in the media, an inhibitor of the HIS3-encoded dehydratase, can reduce Bait-mediated background trans-activation to workable levels. 13 2. The Bait is proteolytically cleaved or is expressed at low levels: If Bait is predicted to be - 6 0 - 8 0 kDa or larger and/or is expressed at low levels, a truncated fusion protein approach may be the best strategy (LexA system I or II).

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