Published: Vol 7, Iss 12, Jun 20, 2017 DOI: 10.21769/BioProtoc.2348 Views: 9085
Reviewed by: Arsalan DaudiVasudevan AchuthanPooja Saxena
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Abstract
This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG 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: ImmunoprecipitationBackground
Immunoprecipitation followed by mass spectrometry (IP/MS) is an unbiased method to identify protein-protein interactions with a specific bait protein of interest. While this approach has been fruitfully applied to identify protein interaction networks, it is plagued by false positives–proteins that appear to interact but are actually non-specifically bound to the beads or antibodies used in the affinity purification. In particular, highly abundant proteins such as ribosomal proteins, metabolic enzymes and chaperone proteins are common contaminants. However, sometimes these common contaminants may be bona fide interaction partners, yet it is challenging to demonstrate specificity. To overcome this obstacle, we developed a serial affinity purification approach to isolate specific, high affinity complexes between bait proteins of interest tagged with two affinity epitopes–the 3xFLAG and V5 tags (Figure 1). We have generated a plasmid containing the 3xFLAG-V5 epitopes and a HIS3 selectable marker that can be amplified and used to C-terminally tag any yeast protein of interest in a single yeast transformation (Zheng et al., 2016). We originally applied our method to demonstrate a specific interaction between the heat shock transcription factor (Hsf1) and the major Hsp70 chaperone proteins present in the yeast cytosol, Ssa1/2. However, this approach can be generally applied to identify high affinity complexes involving a protein of interest. While this technique removes the bulk of false positive interactions, a major caveat is that low affinity and transient interactions are likely to be lost.
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.
Materials and Reagents
Equipment
Procedure
Data analysis
Mass spectrometry data should be analyzed by first ensuring that you observed your bait protein with reasonable coverage (> 25%). Putative interacting proteins should be validated by repeated experiments and alternative detection methods, such as Western blotting (Figure 2). For examples of peptide counts of a bait protein, binding partners and nonspecific contaminants, see Figure 1–source data 1 in Zheng et al., 2016.
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).
Notes
Despite the stringency of the serial affinity protocol, background contaminants will still be observed in the mass spectrometry data. In general, the repertoire of highly abundant ribosomal proteins and metabolic enzymes that are nonspecific contaminants is not very reproducible, though a subset is always present. Thus, with enough replicates, most of these contaminants can be discarded.
Recipes
Acknowledgments
This protocol was adapted from our previous work (Zheng et al., 2016). This work was supported by a grant from the Office of the Director of the National Institutes of Health to D.P. (DP5 OD017941-01).
References
Article Information
Copyright
Zheng and Pincus. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Category
Microbiology > Microbial cell biology > Cell-based analysis
Biochemistry > Protein > Interaction
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