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Membrane System Yeast Library Construction to study interactions of membrane proteins (CAT#: STEM-MB-0014-WXH)

Introduction

Yeast libraries are obtained by extracting the total RNA of the studied species, then isolating the mRNA and obtaining cDNA by reverse transcription, after which we ligate these cDNAs with AD vectors to obtain an AD library, which is then transferred into yeast, and theoretically, such a yeast library is capable of expressing all the proteins of the studied species.<br />The DUAL membrane system (DUAL membrane system) is a membrane protein yeast two-hybrid system based on isolated split-ubiquitin-mediated protein analysis. It provides an in vivo analysis of proteins different from the conventional yeast two-hybrid system, making the analysis of interactions between membrane proteins a reality. Its main feature is that it can perform interactions between membrane protein-membrane protein and membrane protein-soluble protein.<br />Ubiquitin can be divided into two parts: N-terminal (Nub) and C-terminal (Cub). Nub has a strong affinity with Cub, and when expressed simultaneously in the same cell, they will bind to form an intact ubiquitin, which will be recognized and cleaved by ubiquitin proteases (UBPs). The cleaved transcription factor LexA-VP16 enters the nucleus and initiates the expression of reporter genes.




Applications

1.Main application: Detecting the interactions of membrane proteins
2.Other applications:
• Plants: study of self-incompatibility mechanism
• Viruses: prion and viral protein interaction protein screening
• Signaling pathways: transcription factors, estrogen alpha receptor interaction protein screening
• Cancer medicine: cancer-related proteins, apoptosis-related protein interaction protein screening
• Parasitic medicine: virulence factor pathogenesis study, galactose lectin interaction protein screening
Fundamental principles research: myosin, proteasome regulator interaction protein screening, fertilized egg development

Procedure

1.Total RNA extraction
2.mRNA purification and reverse transcription
3.Double-stranded cDNA synthesis and purification
4.Co-transfer to yeast receptor cells
5.Library potency determination

Notes

Customer provides tissue, cells or total RNA