Recognition Imaging on Native Chromatin Extracted from Cancerous and Non-Cancerous Cell Lines

Abstract

Atomic force microscopy (AFM) recognition imaging is a label-free technique which can identify specific molecules from compositionally complex samples while mapping the topography of the samples simultaneously in a liquid environment. It required a sensitive imaging mode. Typically, the so-called MAC mode is used, where the AFM tip is driven directly by a magnetic field which will reduce the movement of liquid surrounding the tip and increase signal to noise ratio. The AFM tips for MAC mode need to have a ferromagnetic coating like nickel in order to be driven by a magnetic field. Commercial MAC mode probes are typically silicon nitride or quartz. We developed a protocol to coat sharp commercial silicon probes with nickel, making them reliable MAC mode tips. We applied our new probes to samples prepared from the supernatant fraction of a nuclear extract of EPC2 (non-cancerous) and CPD (cancerous) esophageal cell lines. Salt fractionation is an efficient method for the extraction of intact chromatin fragments from cell nuclei, but the supernatant fraction was not studied in previous work. With anti-H3 antibody modified MAC mode tips, we identified histones H3 in samples of both cell lines, indicating that this fraction contains chromatin. We proved the specificity by blocking with a peptide mimicking the H3 binding site for our antibody. Further, we demonstrated the dependence of the recognition signals on the oscillation amplitude of the probe, indicating the robustness of our recognition signal. We also analyzed the nuclear extracts for the presence of SMC2, a structural maintenance protein for chromosomes. We applied AFM recognition imaging with anti-SMC2 antibody modified probes to the prepared chromatin samples and identified SMC2 proteins in samples of both cell lines. Western blots confirmed the presence of SMC2 proteins in the samples. Overall, AFM recognition imaging provides a robust way to analyze the structure and molecular components of nuclear extracts from human cells, providing insights into the link between chromatin structure and cell phenotypes.