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Dynamic Measurement of Proteins in Multiple Complexes

Most protein complexes are characterized outside of their native environment as a result of lysing the cell and pulling down the target complex via immunoaffinity.  While this scheme provides valuable data, it does not always provide a specific picture of the protein’s functional role within the cell.  Also since many proteins have multiple functions it is important to be able to characterize its function at a specific time and place within the cell.  The project would directly benefit the many NCI PIs that are interested in studying the protein complex formation associated with their protein of interest.  The aim is to move from providing a list interacting proteins to describing the interaction in much more details with the goal of helping delineating the role of the protein in cancer etiology at the molecular level.

Scientists within the Laboratory of Proteomics and Analytical Technologies (LPAT) are developing methods using Foster resonance energy transfer (FRET) and Duolink (Bioscience) to study the protein composition of different protein complexes that both harbor the same protein of interest.  These two methods compliment each other well allowing for probing of the proteins in both tagged and native configuration and in live and fixed cells.  Our initial experiments are focused on using these two methods to study specific chromosomal location and the composition of protein complexes.  This will be done in the context of different physiological conditions such as apoptosis versus growth promotion and the change in chromosomal location of the complex composition evaluated.

Proximity Ligation Assay The Proximity Ligation Assay (PLA, Duolink II system) from Olink Biosciences was used to visualize the interaction of methyl CpG binding protein 3 (Mbd3) and histone deacetylase 1 (HDAC1). The interaction between Mbd3 and HDAC1 (red) in the Ntera2 cell line is shown. The image demonstrates the localization of the Mbd3/HDAC1 complex with the nucleus (DAPI, blue) in resting cells (*, asterisk).

Page last updated July 2, 2014 @ 4:27 pm