Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins
نویسندگان
چکیده
This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3×FLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3×FLAG antigen peptide. Next, the bait protein is recaptured from the first eluate by anti-V5-conjugated magnetic beads and eluted with ionic detergent. In this manner, the majority of abundant, nonspecific proteins remain either bound to the first beads or in the first eluate, allowing pure, high affinity complexes to be obtained. This approach can be used to show specific interactions with notoriously 'sticky' chaperone proteins.
منابع مشابه
Stability of Recombinant Proteins in Escherichia coli: The Effect of Co-Expression of Five Different Chaperone Sets
Chaperones are produced by prokaryotic, yeast and higher eukaryotic cells for various purposes. Over-expression of each chaperone or sets of them affect the production level of a recombinant protein in the cell. On the basis of this hypothesis, five different plasmids with 5 different combinations of 6 chaperones molecule, transformed into Escherichia coli along with human basic Fibroblast Grow...
متن کاملExpanding the repertoire of plasmids for PCR-mediated epitope tagging in yeast.
Epitope tagging of yeast proteins provides a convenient means of tracking proteins of interest in Western blots and immunoprecipitation experiments without the need to raise and test specific antibodies. We have constructed four plasmids for use as templates in PCR-based epitope tagging in the yeast Saccharomyces cerevisiae. These plasmids expand the range of epitopes available in a tag-URA3-ta...
متن کاملA Combination Method of Centrality Measures and Biological Properties to Improve Detection of Protein Complexes in Weighted PPI Networks
Introduction: In protein-protein interaction networks (PPINs), a complex is a group of proteins that allows a biological process to take place. The correct identification of complexes can help better understanding of the function of cells used for therapeutic purposes, such as drug discoveries. One of the common methods for identifying complexes in the PPINs is clustering, but this study aimed ...
متن کاملA Combination Method of Centrality Measures and Biological Properties to Improve Detection of Protein Complexes in Weighted PPI Networks
Introduction: In protein-protein interaction networks (PPINs), a complex is a group of proteins that allows a biological process to take place. The correct identification of complexes can help better understanding of the function of cells used for therapeutic purposes, such as drug discoveries. One of the common methods for identifying complexes in the PPINs is clustering, but this study aimed ...
متن کاملRole of Molecular Interactions and Oligomerization in Chaperone Activity of Recombinant Acr from Mycobacterium tuberculosis
Background: The chaperone activity of Mycobacterium tuberculosis Acr is an important function that helps to prevent misfolding of protein substrates inside the host, especially in conditions of hypoxia. Objectives: The aim of this study was to establish the correlation of structure and function of recombinant Acr proteins both before and after ge...
متن کامل