Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins

Abstract

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.

Keywords: FLAG tag; Immunoprecipitation; Protein complexes; V5 tag; Yeast.

Figure 1.. Schematic overview of the protocol.

A 3xFLAG/V5 dual-tagged bait protein is serially purified with anti-FLAG beads followed by anti-V5 beads.

Figure 2.. Western blot of immuno-precipitated Hsf1-3xFLAG/V5.

3xFLAG/V5-tagged Hsf1 was serially purified with anti-FLAG and anti-V5 beads from cells under control (-) and heat shock conditions (+). Hsf1 is low abundance and cannot be easily detected in the input, but is enriched following immunoprecipitation. Hsf1 migrates slower in the gel under heat shock conditions due to phosphorylation (HS = heat shock).