Assembling and imaging of his‐tag green fluorescent protein on mica surfaces studied by atomic force microscopy and fluorescence microscopy

Abstract

The adsorption of his-tag green fluorescent protein (GFPH(6)) onto the mica surfaces has been studied by atomic force microscopy (AFM) and laser confocal fluorescence microscopy. By controlling the adsorption conditions, separated single GFPH(6) and GFPH(6) monolayer can be adsorbed and formed on mica surfaces. In present experiments, based on the AFM measurement, we found that the adsorbed GFPH(6) was bound on the mica surface with its beta-sheets. The formed GFPH(6) monolayer on mica surfaces was flat, uniform, and stable. Some applications of the formed monolayer have been demonstrated. The formed monolayer can be used as a substrate for DNA imaging and AFM mechanical lithography.

Adsorption of human serum albumin onto highly orientated pyrolytic graphite surface studied by atomic force microscopy

“…The actual dimension of HSA should be less than these values due to the well‐known broadening effect caused by the finite probe tip radius in AFM imaging. The actual dimension of HSA can be estimated by a previously proposed formula to deconvolute the tip effects (Liu et al ., ). The calculated length of HSA after deconvolution is 12.6 nm and the width is 6.5 nm when the AFM tip radius assumed to be 10 nm.…”

Imaging and studying human topoisomerase I on mica surfaces in air and in liquid by atomic force microscopy

“…When the size of the observed samples are equivalent to or smaller than the curvature radius of AFM tip, the well-known broadening effects arise (Bustamante et al 1992), which induced the observed length and width to be larger than its true values. This phenomenon has been verified in green fluorescent protein imaging by AFM in liquid (Liu et al 2008). Unfortunately, the central DNA-binding pore of TOPO I cannot be The observed longest part of a TOPO I was measured as its length; the height and the width was measured at the vertical center according to its length, and the width was measured at half maximum height.…”

“…TOPO I monolayer can be formed by adsorbing high concentration of TOPO I on mica surface. The TOPO I monolayer on mica surface has very stable characteristics, and may have some potential applications (Liu et al 2008).…”

“…Especially for the assembly consisting of many different proteins or some large biomolecules with difficulty in obtaining their crystals or single protein adsorbed on a solid surface that is difficult to be studied by the X-ray diffraction technique, AFM can still be utilized to obtain three-dimensional structures at high resolution (Bahatyrova et al 2004). Furthermore, the three-dimensional structures of some biomolecules obtained by these two techniques can be compared and verified with each other (Kim et al 2002;Liu et al 2008). Thus, AFM can be regarded as a complementary technique to X-ray diffraction providing structural information about biomacromolecules (Fotiadis et al 2002).…”

“…For studying the topography and the structures of biomacromolecules on a substrate, atomic force microscopy (AFM) is a very useful tool. A single molecule such as DNA or protein has been studied by AFM (Fotiadis et al 2002;Kobayashi et al 2007;Kuznetsov and Mcpherson 2006;Liu et al 2005;Liu et al 2008;Quist et al 2005;Zhang et al 2009). Especially for the assembly consisting of many different proteins or some large biomolecules with difficulty in obtaining their crystals or single protein adsorbed on a solid surface that is difficult to be studied by the X-ray diffraction technique, AFM can still be utilized to obtain three-dimensional structures at high resolution (Bahatyrova et al 2004).…”