Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.
Revised: October 16, 2008 |
Published: April 11, 2005
Citation
Markillie L.M., C.T. Lin, J.N. Adkins, D.L. Auberry, E.A. Hill, B.S. Hooker, and P.A. Moore, et al. 2005.Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks.Journal of Proteome Research 4, no. 2:268-274.PNNL-SA-42494.doi:10.1021/pr049847a