Antibody and Protein Purification Unit

Thursday, October 27, 2011 — Core Poster Session
10:00 a.m. – Noon	South Lobby of Building 10	NCI	CORE-11

Authors

• C Heger
• J Chen
• C McAndrew
• M Herrmann
• P Goldsmith

Abstract

The Antibody and Protein Purification Unit core facility within the CCR has evaluated new ultrasensitive antibody based technologies using cancer cell line and tissue derived source materials. Our validated technologies include: NanoPro capillary isoelectric focusing immunoassays (Cell Biosciences) for sensitive detection of proteins and their phosphoisoforms; In situ proximity ligation assays (PLA, Olink) for detection and quantization of protein interactions and low abundance proteins in cells and tissues; and Solution-phase PLA (ABI) for two-site assays capable of detecting ligands using standard curves spanning one to three hundred cells. Each of these technologies, which require extremely small amounts of assay materials, has been directed towards translational studies using patient samples. Using the NanoPro, we can detect phosphorylation of members of the MAP kinase family (Erk1/2, phospho-Erk1/2, Mek1, phosphoMek1); protein kinase C isoforms; other signaling molecules including phospho-Jnk and Akt (pan-Akt 1/2/3 and various phosphoisoforms). The in situ PLA method has allowed us to look at receptor dimerization events (Her2/EGFR heterodimers; Prolactin receptor homo and heterodimers) and stimulus-induced protein phosphorylations (phospho-PDGFR, phospho-Cmet, phospho-EGFR) without the need to epitope-tag or modify the cells or tissue. Solution-phase PLA has been used to generate sensitive assays for the detection of Mek1/pMek1, Prolactin, C-Met, and Osteopontin.

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