Tandem Affinity Purification Assay to Study Protein-Protein Interactions

In dit artikel

Samenvatting

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 levels and solubility of the protein complex of interest. Alternatively, if larger-scale purification is needed, cell lines can be adapted to grow in suspens....

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Resultaten

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 -.......