Glutaraldehyde modified mica: a new surface for atomic force microscopy of chromatin.
We have found that mica surfaces functionalized with aminopropyltriethoxysilane and aldehydes bind chromatin strongly enough to permit stable and reliable solution imaging by atomic force microscopy. The method is highly reproducible, uses very small amounts of material, and is successful even with very light degrees of surface modification. This surface is far superior to the widely used aminopropyltriethoxysilane-derivatized mica surface and permits resolution of structure on the nanometer-scale in an aqueous environment, conditions that are particularly important for chromatin studies. For example, bound nucleosomal arrays demonstrate major structural changes in response to changes in solution conditions, despite their prior fixation (to maintain nucleosome loading) and tethering to the surface with glutaraldehyde. By following individual molecules through a salt titration in a flow-through cell, one can observe significant changes in apparent nucleosome size at lower [salt] and complete loss of DNA from the polynucleosomal array at high salt. The latter result demonstrates that the DNA component in these arrays is not constrained by the tethering. The former result is consistent with the salt-induced loss of histones observed in bulk solution studies of chromatin and demonstrates that even histone components of the nucleosome are somewhat labile in these fixed and tethered arrays. We foresee many important applications for this surface in future atomic force microscopy studies of chromatin.
Wang
Hongda
H
Department of Physics and Astronomy, Arizona State University, Tempe, AZ 85287, USA.
Bash
Ralph
R
Yodh
Jiya G
JG
Hager
Gordon L
GL
Lohr
D
D
Lindsay
Stuart M
SM
D000538:Aluminum Silicates / Q000737:chemistry*; D002843:Chromatin / Q000737:chemistry* / Q000648:ultrastructure*; D005976:Glutaral / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods*; D009707:Nucleosomes / Q000737:chemistry / Q000648:ultrastructure; D011786:Quality Control; D012965:Sodium Chloride / Q000737:chemistry; D012996:Solutions / Q000737:chemistry; D013499:Surface Properties
D000538:Aluminum Silicates; D002843:Chromatin; D009707:Nucleosomes; D012996:Solutions; D012965:Sodium Chloride; D005976:Glutaral; C011934:mica