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. 2015 May 14;26(6):975–986. doi: 10.1021/acs.bioconjchem.5b00191

Figure 4.

Figure 4

Pull-down assays for the discovery of protein complexes. (A) Schematic illustration of HaloTag pull-down assays, in which a single HaloTag construct encoding a bait protein is stably transfected into a cell line. The bait protein interacts with additional proteins, at which time cells are lysed and captured using HaloLink resin. Pure proteins can be eluted using a detergent (e.g., SDS), or protein complexes attached the bait protein can be eluted using TEV cleavage. Reprinted with permission from ref (57). Copyright 2014 JoVE. (B) A pull-down assay was performed using HaloTag-modified RpoA to determine the efficiency of HaloTag to extract multiprotein complexes. M, molecular weight marker; 1, unbound proteins; 2, washed proteins; 3, eluted proteins after TEV cleavage; 4, eluted proteins after removal of TEV; and 5, concentrated protein sample. Arrows indicate recombinant HaloTag in lane 1 and cleaved RpoA in lane 3. In addition, a protein interaction map was constructed from data using MALDI-MS/MS. Reprinted with permission from ref (61). Copyright 2012 Peterson and Kwon.