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Abstract

Fast fixation is necessary to study real time protein-protein interactions under physiological conditions. Fast formaldehyde cross linking can fix transient and weak protein interactions, thereby reducing the number of false negatives but producing great complexity. To reduce this complexity, immunoaffinity purification can fish out complexes that include particular target proteins, but affinity based co-purification has a limited capacity to eliminate non specific binding to beads and/or antibodies. To filter out these complexes, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncross linked complexes and simultaneously providing molecular weight information for identification. We described a 4F-acts strategy to help improve real time ligands discovery based on formaldehyde cross linking, immunoprecipitation and SDS-PAGE separation: Fast Fix, Fish, and Filter, using albumin interactome as an example. The use of gel excision without staining makes this strategy comprehensive and sensitive. The target protein must be identified in the same slice as its ligands. The ligands must be identified in slices for the experimental group but not in the corresponding control slices. Only proteins that appear in the range of molecular weights equal to or greater than the sum of the proteins� theoretical molecular weights, together with the target are considered ligands. In this study, 5 s of cross linking with 10% formaldehyde was achieved in human blood. The use of this strategy identified 35 ligands for albumin. Comparison with four major previous studies of the albuminome revealed that 68.57% of the 35 ligands identified in our study were identified in these other studies. Fast cross linking was achieved. The 4F-acts strategy can be used to identify real time in situ interactions without prior intervention and to comprehensively identify ligands of particular target proteins with fewer false positives.

Biography

Youhe Gao is the Professor at Beijing Normal University, China. He has received his MD from Peking Union Medical College, PhD from University of Connecticut and Postdoctoral training from Beth Israel Deaconess Medical Center Harvard Medical School. He was the Professor of Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/Peking Union Medical College from 2001-2014. His research interests include biomarker discovery in urine proteome, protein interaction and related bioinformatics.

Email: gaoyouhe@bnu.edu.cn