Capturing protein-protein complexes at equilibrium: the holdup comparative chromatographic retention assay.
The popular pulldown chromatographic assay detects complexes mediated by fusion proteins retained on affinity resin. The main limitation of this method is that it does not analyze complexes at equilibrium but after several washing steps. Consequently, fast-dissociating complexes may remain undetected. Here, we present the holdup assay, based on the principle of comparative chromatographic retention which eliminates the use of washing steps. The assay evaluates fractions of free and bound species at equilibrium. We used human papillomavirus oncoprotein E6, an E6-binding peptide and an E6-binding PDZ domain, to test several protocols utilizing pure proteins or expression extracts. The holdup assay is faster and more informative than the pulldown assay. It detects fast-dissociating complexes and it is also suited for evaluating equilibrium constants. It is potentially adaptable for automated determination of affinity constants and high-throughput analysis of interactions between proteins and other proteins, peptides, nucleic acids, or small regulatory molecules.
Charbonnier
Sebastian
S
Equipe Oncoprotéines, UMR CNRS 7175-LC1, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France.
Zanier
Katia
K
Masson
Murielle
M
Travé
Gilles
G
D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods*; D005982:Glutathione Transferase / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012117:Resins, Synthetic / Q000737:chemistry; D012680:Sensitivity and Specificity
C068651:E6 protein, Human papillomavirus type 16; D008024:Ligands; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D012117:Resins, Synthetic; D005982:Glutathione Transferase