Nuclear factor-kB (NF-kB) 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-kB 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-kB DNA binding activity. NF-kB target sequence DNA has been employed to mica functionalization in order to set up a surface able to capture transcriptionally active NF-kB protein complexes from cell lysates, with the aim to detect DNA binding capacity of NF-kB 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-kB at pico- Molar proteins concentration range from cultured cell samples and from biological fluid cells permitting us to estimate NF-kB 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-kB based bio-diagnostic nano-device.

Label-free quantification of activated NF-kB in biological samples by atomic force microscopy

MENOTTA, MICHELE;CRINELLI, RITA;CARLONI, ELISA;BIANCHI, MARZIA;GIACOMINI, ELISA;MAGNANI, MAURO
2010-01-01

Abstract

Nuclear factor-kB (NF-kB) 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-kB 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-kB DNA binding activity. NF-kB target sequence DNA has been employed to mica functionalization in order to set up a surface able to capture transcriptionally active NF-kB protein complexes from cell lysates, with the aim to detect DNA binding capacity of NF-kB 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-kB at pico- Molar proteins concentration range from cultured cell samples and from biological fluid cells permitting us to estimate NF-kB 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-kB based bio-diagnostic nano-device.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11576/2504201
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