title: Label-free quantification of activated NF-kappaB in biological samples by atomic force microscopy.
abstract: Nuclear factor-kappaB (NF-kappaB) is a ubiquitous transcription factor involved in the pro-inflammatory response to several factor, and in auto-inflammatory diseases. The usual methods for detection of NF-kappaB DNA binding activity are the electrophoretic mobility shift assay (EMSA), and enzyme-linked immunosorbent assay (ELISA). Here we report a development of a quantitative atomic force microscopy (AFM) based technique, for the analysis of NF-kappaB DNA binding activity. NF-kappaB target sequence DNA has been employed to mica functionalization in order to set up a surface able to capture transcriptionally active NF-kappaB protein complexes from cell lysates, with the aim to detect DNA binding capacity of NF-kappaB from low amount of biological samples such as biopsy. We were able to obtain images of the captured complex on the surface and furthermore we carried out an AFM images quantification. We were able to quantify relative and absolute quantities of NF-kappaB at pico-Molar proteins concentration range from cultured cell samples and from biological fluid cells permitting us to estimate NF-kappaB binding activity. The results obtained by AFM imaging have been compared and validated with EMSA. The present work represents the first quantification approach by AFM analysis. The results and the method may be used toward development of NF-kappaB based bio-diagnostic nano-device.
authors.affiliation: Department of Biomolecular Sciences, University of Urbino Carlo Bo Via Saffi 2 61029 Urbino, PU, Italy. michele.menotta@uniurb.it
authors.forename: Michele
authors.initials: M
authors.lastname: Menotta
authors.forename: Rita
authors.initials: R
authors.lastname: Crinelli
authors.forename: Elisa
authors.initials: E
authors.lastname: Carloni
authors.forename: Marzia
authors.initials: M
authors.lastname: Bianchi
authors.forename: Elisa
authors.initials: E
authors.lastname: Giacomini
authors.forename: Ugo
authors.initials: U
authors.lastname: Valbusa
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
mesh_terms: D004247:DNA / Q000032:analysis* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008264:Macrophages / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D016328:NF-kappa B / Q000032:analysis* / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D016328:NF-kappa B; D004247:DNA