Seven hundred base pair regions from ompC (−607 to +93 bp), cadB (−673 to +27 bp), and yghA (−673 to +27 bp) were gel-purified using the QIAquick Gel Extraction Kit (Qiagen). A glutaraldehyde-modified mica surface was prepared as described46 (link). Ten nanogram of the regulatory region was incubated with 30 nM OmpR or OmpR~P (prepared by phosphorylation from acetyl phosphate35 (link) at pH 5.6 or 7.2 ± 15% (w/v) sucrose for 15 min at RT. This mixture was then deposited on the mica for 15 min. Images were acquired on a Bruker Dimension FastScan AFM system using the tapping mode with a silicon nitride cantilever (FastScan C, Bruker). Raw AFM images were processed using Gwyddion software (http://gwyddion.net/ ).
Fastscan c
Manufactured by Bruker
Sourced in Morocco, United States
The FastScan C is a high-speed atomic force microscopy (AFM) system designed for advanced nanocharacterization. It provides fast and precise topographical imaging of samples at the nanoscale level.
Automatically generated - may contain errors
8 protocols using fastscan c
1
Atomic Force Microscopy of DNA-Protein Interactions
2
Visualizing DNA-Protein Interactions on Mica
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Glutaraldehyde-modified mica surface was prepared as decribed previously in (Liu et al., 2010 (link)) and references therein. A 755 bp sequence upstream to the +1 start site (Gerstel et al., 2003 (link)) of csgD was amplified and gel purified. This fragment also harbored a SsrB-specific site (Feng et al., 2004 (link); Walthers et al., 2007 (link)), TTATAAT sequence (Figure 6—figure supplement 4 ). A typical 50 μl reaction contained 10 ng of this DNA (755 bp of the csgD regulatory region) mixed with an appropriate amount of SsrB or H-NS and incubated for 15 min at room temperature. This mixture was then deposited on glutaraldehyde-modified mica for 15 min. Images were acquired on a Bruker Dimension FastScan AFM system using the tapping mode with a silicon nitride cantilever (FastScan C, Bruker). Raw AFM images were processed using Gwyddion software.
3
Atomic Force Microscopy Analysis of gltA Promoter
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A 703 base pair (bp) region from the gltA promoter was gel-purified using the QIAquick Gel Extraction Kit (Qiagen) by using primer pairs gltA F and gltA R for S. Typhimurium (-689 bp to + 14 bp) and for E. coli (-694 bp to + 9 bp), respectively. A glutaraldehyde-modified mica surface was prepared as described in Chakraborty et al. (2017) (link). Ten nanograms of the gltA regulatory region was incubated with 30 nM OmpR for 15 min at RT. This mixture was then deposited on the mica for 15 min. Images were acquired on a Bruker Dimension FastScan AFM system using the tapping mode with a silicon nitride cantilever (FastScan C, Bruker). Raw AFM images were processed using Gwyddion software1 .
4
AFM Nanomechanical Mapping Technique
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AFM
measurements were performed with a Bio FastScan scanning probe microscope
(Bruker AXS). All images were obtained using the PeakForce QNM (PeakForce
quantitative nanomechanical mapping) mode with a FastScan C (Bruker)
silicon probe (spring constant of 0.45 N/m).
The measurements
were performed under environmental conditions in the acoustic hood
to minimize vibrational noise. The images were captured in the retrace
direction at a scan rate of 1.6 Hz. The image resolution was 512 samples/line.
For image processing and thickness analysis, Nanoscope Analysis software
was used. The “flattening” and “planefit”
functions were applied to each image.
measurements were performed with a Bio FastScan scanning probe microscope
(Bruker AXS). All images were obtained using the PeakForce QNM (PeakForce
quantitative nanomechanical mapping) mode with a FastScan C (Bruker)
silicon probe (spring constant of 0.45 N/m).
The measurements
were performed under environmental conditions in the acoustic hood
to minimize vibrational noise. The images were captured in the retrace
direction at a scan rate of 1.6 Hz. The image resolution was 512 samples/line.
For image processing and thickness analysis, Nanoscope Analysis software
was used. The “flattening” and “planefit”
functions were applied to each image.
5
Nanoscale Peptide Imaging on Graphite
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All peptides were purchased from Genscript (http://www.genscript.com/ ) at >98% purity (HPLC purified), and all buffers were prepared in ultrapure deionized water (Millipore, Billerica, MA). For pH study, a tricomponent buffer composed of citrate, HEPES, and CHES was used (broad-range buffer CHC; Molecular Dimensions, Altamonte Springs, FL). Highly oriented pyrolytic graphite (HOPG) slabs for imaging on graphite were purchased from SPI Supplies (Structure Probe, West Chester, PA). Atomic force microscopy (AFM) imaging was performed with a FastScan AFM instrument (Bruker Instruments, Billerica, MA) using soft triangular-shaped silicon nitride cantilevers (FastScan C, Bruker Instruments) characterized by a nominal spring constant of k ∼ 0.8 N/m and a nominal resonant frequency of 300 kHz. Imaging was performed using the FastScan device’s ScanAsyst Mode at speeds of ∼4 lines/s for optimal topographic quality. For AFM imaging under fluid, we used a home-made perfusion cell that allowed the fluid medium to be refreshed.
6
Quantitative Nanomechanical Mapping by AFM
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AFM measurements were carried out using a Bio FastScan scanning probe microscope (Bruker AXS) under environmental conditions in the acoustic hood to minimize vibrational noise. Images were obtained by applying the PeakForce quantitative nanomechanical map-ping (QNM) mode with a FastScan-C (Bruker) silicon probe (spring constant of 0.45 N/m) in the retrace direction with a scan rate of 1.7 Hz (512 samples/line). Image processing and roughness analysis were performed using the Nanoscope Analysis 1.5 software by applying the "flatting" and "planefit" functions.
7
Binding Dynamics of SsrB on csgD Promoter
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Glutaraldehyde-modified mica surface was prepared as decribed previously in (Liu et al., 2010 (link)) and references therein. A 755 bp sequence upstream to the +1 start site (Gerstel et al., 2003 (link)) of csgD was amplified and gel purified. This fragment also harbored a SsrB-specific site (Feng et al., 2004 (link); Walthers et al., 2007 (link)), TTATAAT sequence (Figure 6—figure supplement 4 ). A typical 50 μl reaction contained 10 ng of this DNA (755 bp of the csgD regulatory region) mixed with an appropriate amount of SsrB, SsrBc or D56A SsrB and incubated for 15 min at room temperature. This mixture was then deposited on glutaraldehyde-modified mica for 15 min. Images were acquired on a Bruker Dimension FastScan AFM system using the tapping mode with a silicon nitride cantilever (FastScan C, Bruker). Raw AFM images were processed using Gwyddion software (http://gwyddion.net/ ). The bending angle was analysed using a home-written Matlab code as described in the Supplementary methods.
8
Lipid Sample Characterization by AFM
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Lipid samples on mica were characterized by PeakForce Tapping mode AFM using a Dimension FastScan (Bruker, USA). All scans were performed in the imaging buffer using ultrasharp silicon tip cantilevers (FastScan-C, Bruker, USA) with a 0.8 N m−1 force constant and 5 nm tip radius.
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