Tandem Affinity Purification Assay to Study Protein-Protein Interactions

Abstract

Source: Ma, Z., et al. Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells. J. Vis. Exp. (2017).

This video demonstrates tandem affinity purification — a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.

Protocol

1. Plating Cells One day prior to transfection, plate 3.5 x 106 HEK293T cells in 10 mL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/Streptomycin (10,000 U/ml stock) per 100 mm dish. Plate 3 – 5 100 mm2 dishes per experiment/condition to ensure sufficient protein recovery. NOTE: The number of plates has to be determined for each experiment/condition, which will rely on the expression lev…

Representative Results

Table 1: Plasmids used in this Study.  CycT1 and CDK9 were ligated into the pcDNA/4TO-STREP and pcDNA/4TO-FLAG plasmid vectors. These constructs were transformed into DH5α competent cells and plated on LB-Ampicillin plates. Colonies were selected, grown, and screened. Successfully ligated clones were verified by Sanger sequencing and the combination of restriction digestion and agarose gel electrophoresis. For CycT1 we used a shorter version (1 – 708) instead of the full-length (1 -…

Materials

Dulbecco's Modified Eagle Medium (DMEM)	Fisher Scientific	SH30022FS
Fetal bovine serum	Hyclone	SH30071
Penicillin/Streptomycin	Fisher Scientific	MP091670049
PolyJet	Fisher Scientific	50-478-8
Protease inhibitor cocktail	Roche	11836153001
STREP-Tactin Superflow	IBA	2-1208-010
STREP-tag elution buffer	IBA	2-1000-025
EZview Red ANTI-FLAG M2 Affinity Gel	Sigma	SLBQ1981V
Corning 100mm × 20 mm style Dish cell culture treated nonpyrogenic polystyrene 20/sleeve	Corning	430167
Protein Lo-Bind Eppendorf	Fisher Scientific	13-698-794
Digital Vortex Mixer	Fisher Scientific	02-215-370
48-hole micro tube foam rack	Fisher Scientific	02-215-386
Labquake shaker rotisserie	Thermo	415110