Label-free quantification of activated NF-kappaB in biological samples by atomic force microscopy.
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.
Menotta
Michele
M
Department of Biomolecular Sciences, University of Urbino Carlo Bo Via Saffi 2 61029 Urbino, PU, Italy. michele.menotta@uniurb.it
Crinelli
Rita
R
Carloni
Elisa
E
Bianchi
Marzia
M
Giacomini
Elisa
E
Valbusa
Ugo
U
Magnani
Mauro
M
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
D016328:NF-kappa B; D004247:DNA