Atomic force microscopy imaging and probing of DNA, proteins, and protein DNA complexes: silatrane surface chemistry.
Despite their rather recent invention, atomic force microscopes are widely available commercially. AFM and its special modifications (tapping mode and noncontact operation in solution) have been successfully used for topographic studies of a large number of biological objects including DNA, RNA, proteins, cell membranes, and even whole cells. AFM was also successfully applied to studies of nucleic acids and various protein DNA complexes. Part of this success is due to the development of reliable sample preparation procedures. This chapter describes one of the approaches based on chemical functionalization of mica surface with 1-(3-aminopropyl) silatrane (APS). One of the most important properties of APS-mica approach is that the sample can be deposited on the surface in a wide range of ionic strengths, in the absence of divalent cations and a broad range of pH. In addition to imaging of dried sample, APS-mica allows reliable and reproducible time lapse imaging in aqueous solutions. Finally, APS mica is terminated with reactive amino groups that can be used for covalent and ionic attachment of molecules for AFM force spectroscopy studies. The protocols for the preparation of APS, functionalization with APS mica and AFM probes, preparation of samples for imaging in air and in aqueous solutions, and force spectroscopy studies are outlined. All these applications are illustrated with a few examples.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA.
Yuri L
YL
Lyubchenko
Luda S
LS
Shlyakhtenko
Alexander A
AA
Gall
D000388:Air; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000138:chemical synthesis / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004278:DNA, Superhelical / Q000648:ultrastructure; D018625:Microscopy, Atomic Force / Q000379:methods*; D017646:Organosilicon Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012996:Solutions; D013057:Spectrum Analysis; D013499:Surface Properties; D013997:Time Factors; D014867:Water; D051844:alpha-Synuclein / Q000378:metabolism
D019086:Bridged Bicyclo Compounds, Heterocyclic; D004278:DNA, Superhelical; D017646:Organosilicon Compounds; D011506:Proteins; D012996:Solutions; D051844:alpha-Synuclein; D014867:Water; C015503:silatrane; D011092:Polyethylene Glycols; D004247:DNA