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Jacak J.,Johannes Kepler University | Schnidar H.,University of Salzburg | Schnidar H.,Biologics | Muresan L.,Johannes Kepler University | And 6 more authors.
Journal of Biotechnology | Year: 2013

We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138neg) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138neg cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material. © 2013 Elsevier B.V. Source


Zhang J.,CAS Changchun Institute of Applied Chemistry | Zhang J.,University of Chinese Academy of Sciences | Liu H.,CAS Changchun Institute of Applied Chemistry | Liu H.,University of Chinese Academy of Sciences | And 5 more authors.
Analyst | Year: 2013

The understanding of ligand binding interactions is an important component of understanding the fundamental mechanism of receptor function. In this study, the binding abilities of EGF and TGF-α to EGFR on human bladder cancer (T24) cells were investigated by single molecular force spectroscopy (SMFS) based on atomic force microscopy (AFM). By approaching the specifically functionalized AFM tips to the T24 cell surface and subsequent retraction, specific unbinding events of the EGF/EGFR complexes and TGF-α/EGFR complexes were investigated. Further, the unbinding forces and the kinetic off rate constants that govern the bond stabilities were calculated through varying the external mechanic forces applied. Meanwhile, the distances from the energy minimum to the transition states along the separation paths of the EGF/EGFR complexes and TGF-α/EGFR complexes were deduced. This study at single-molecule level may enrich our understanding of the ligand binding properties of EGFR and provide some new information to the development of improved EGFR inhibitors. In addition, the results present new insight into the study of the energy landscape of the dissociation of ligand-EGFR system. © 2013 The Royal Society of Chemistry. Source


Chtcheglova L.A.,Center for Advanced Bioanalysis Gmb
Methods in molecular biology (Clifton, N.J.) | Year: 2013

One of the challenging tasks in molecular cell biology is to identify and localize specific binding sites on biological samples with high spatial accuracy (in order of several nm). During the past 5 years, simultaneous topography and recognition imaging (TREC) has become a powerful AFM-based technique for quick and easy high-resolution receptor mapping. In this chapter, we provide a flavor of TREC application on vascular endothelial cells by describing the detailed procedures for all stages of the experiment from tip and sample preparations through the operating principles and visualization. Source


Chtcheglova L.A.,Center for Advanced Bioanalysis Gmb | Hinterdorfer P.,Center for Advanced Bioanalysis Gmb | Hinterdorfer P.,Johannes Kepler University
Methods in Molecular Biology | Year: 2012

One of the challenging tasks in molecular cell biology is to identify and localize speci fic binding sites on biological samples with high spatial accuracy (in order of several nm). During the past 5 years, simultaneous topography and recognition imaging (TREC) has become a powerful AFM-based technique for quick and easy high-resolution receptor mapping. In this chapter, we provide a flavor of TREC application on vascular endothelial cells by describing the detailed procedures for all stages of the experiment from tip and sample preparations through the operating principles and visualization ©Springer Science+Business Media, LLC 2012. Source


Chtcheglova L.A.,Center for Advanced Bioanalysis Gmb | Hinterdorfer P.,Johannes Kepler University
Methods in Molecular Biology | Year: 2013

The real-time visualization of specific binding sites on biological samples with high spatial resolution, in order of several nanometers, is an important undertaking in many fields of biology. During the past 5 years, simultaneous topography and recognition imaging (TREC) has become a powerful tool to quickly obtain local receptor nanomaps on complex heterogeneous biosurfaces, such as cells and membranes. In this chapter, we present the TREC technique and explain how to unravel the nano-landscape of cells of the immune system, such as macrophages. We describe the procedures for all steps of the experiment including tip functionalization with Fc fragments via flexible PEG-linker, sample preparation, and localization of Fcγ receptors on macrophages. © 2013 Springer Science+Business Media, LLC. Source

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