A protein-protein binding assay using coated microtitre plates: increased throughput, reproducibility and speed compared to bead-based assays.
Protein-protein interactions, and the factors affecting them, are of fundamental importance to all biological systems. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITR) are powerful methods for assaying such interactions, but are expensive to implement. In contrast, bead-based pull-down assays using affinity tags such as glutathione-S-transferase (GST), require no specialist equipment. As a result, such assays are the most popular method for analysing protein-protein interactions, despite being time-consuming and prone to variability. In respect of these problems, we have modified this form of binding assay, using glutathione-coated 96-well plates rather than glutathione-Sepharose beads to bind the primary bait protein. Quantitation of bound protein utilises ELISA for purified proteins and scintillation counting for in vitro translated proteins, rather than the SDS-PAGE-based detection methods used in traditional bead-based assays. These modifications result in an approximately 10-fold increase in the number of samples that can be assayed daily, and allow results to be obtained within hours as opposed to days. We validate the modified assay by analysing the equilibrium binding of Munc18 and syntaxin, and also demonstrate that association and dissociation kinetics may be measured using this approach. The method we describe is generally applicable to any protein-protein interaction assay based on affinity tags and is amenable to automation, and so should benefit a wide range of biochemical research.
Craig
Tim J
TJ
Physiological Laboratory, University of Liverpool, Crown Street, PO Box 147, Liverpool L69 3BX, UK.
Ciufo
Leonora F
LF
Morgan
Alan
A
D001331:Automation; D001671:Biochemistry / Q000379:methods*; D002151:Calorimetry; D002846:Chromatography, Affinity / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors; D033921:Vesicular Transport Proteins / Q000737:chemistry
D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D033921:Vesicular Transport Proteins; D012685:Sepharose; D005982:Glutathione Transferase; D005978:Glutathione