title: Surface plasmon resonance (SPR) spectrometry as a tool to analyze nucleic acid-protein interactions in crude cellular extracts.
abstract: This study presents proof-of-principle application showing that label-free affinity enrichment surface plasmon resonance (SPR) biosensor binding is able to semiquantitatively detect molecular DNA-protein interactions in crude cellular extracts in a real-time ligand fishing analysis study. Crude cell extracts obtained from a confluent HT-28 human adenocarcinoma cell line, synchronized to the G(0)/G(1) phase of the cell cycle, were extracted in a chaotropic medium and cryopreserved in liquid nitrogen. Various immunoprecipitation antibodies were used against defective human excision and mismatch repair genes, hDDB2 and hMSH2, respectively, which theoretically allow for protein binding to DNA ligands in their native conformation. A set of biotinylated DNA target sequence heteroduplexes were also utilized for binding hDDB2 and hMSH2, prepared by heating a biotinylated oligonucleotide strand with an equimolar amount of the complementary strand to form a DNA duplex for hMSH2; a UV-irradiated duplex was employed for hDDB2 instead of an irradiated single-strand DNA to enhance binding. SDS was used to regenerate heteroduplex-modified chips that were used in a BIAcore 2000 SPR instrument at 25°C. Results showed that hMSH2 does not bind preferentially to the heteroduplex-complementary pair. In contrast, hDDB2 was found to bind preferentially to the UV-irradiated version of the heteroduplex-complementary pair. It is concluded that the choice of antibodies with appropriate epitopes is crucial to the success of these SPR binding studies because of enhanced specificity.
authors.affiliation: GEM Tox Consultants & Labs, Inc., 2905 South Memorial Drive, Greenville, NC 27834, U.S.A. gemtoxconsultants@yahoo.com
authors.forename: Farid E
authors.initials: FE
authors.lastname: Ahmed
authors.forename: John E
authors.initials: JE
authors.lastname: Wiley
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Weidner
authors.forename: Chris
authors.initials: C
authors.lastname: Bonnerup
authors.forename: Helvecio
authors.initials: H
authors.lastname: Mota
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation / Q000528:radiation effects; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D045424:Complex Mixtures; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D051718:MutS Homolog 2 Protein / Q000737:chemistry / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
chemical_list: D045424:Complex Mixtures; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein