The Aryl-Hydrocarbon Receptor Protein Interaction Network (AHR-PIN) as Identified by Tandem Affinity Purification (TAP) and Mass Spectrometry
Experimentation overview. (a) Schematic outline of TCDD exposure experiments in Hepa1c1c7 cell lines. GFP and AHR samples were treated with vehicle (DMSO (D), 0.01%); the remaining three AHR samples were treated with TCDD (10 nM) for the labeled time (30, 120, and 240 mins). (b) Schematic of the tandem affinity purification (TAP) protocol. After TCDD exposure, cell lysate samples underwent two rounds of purification utilizing the TAP methodology. AHR and GFP TAP-tagged proteins and their associated complexes were isolated and then separated using gel electrophoresis. (c) Representative image of gel replicates for final TAP eluate samples separated in 4–12% Nu-page gel matrix. Boxes around the areas of the AHR-TAP 240 min TCDD dose sample lane denote the areas of the gel that were excised from each of the sample lanes. (d) Schematic of the in-gel trypsin digestion protocol. Excised gel samples were destained, and proteins underwent reduction, alkylation, and trypsin digestion. (e) Sample of MS analysis data set. Lastly, the protein fragment samples then underwent MS analysis as described in Section 2.
(a) Tissue culture experiment
(b) TAP-tagging method
(c) Protein sample separation
(d) In gel trypsin digestion
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