Proteoliposome stock solutions were diluted to 100 nM Pgp in 20 mM Hepes at pH 7.4, 100 mM NaCl, 5mM MgCl2, which will be called imaging buffer. These solutions were immediately deposited on a freshly cleaved mica surface (Grade v1; Ted Pella, Redding, CA) and incubated for 45 min at ~ 30 °C. During this time, the proteoliposomes rupture and form a planar lipid bilayer supported by the mica surface [32 (link)]. The C-side and EC-side Pgp protein domains protrude out from the bilayer surface making them accessible to AFM imaging. For the antibody experiments, 2 μl of antibody (C219 at 0.07 mg/ml or 6 × His tag antibody at 1 mg/ml) was added to 198 μl of 100 nM Pgp proteoliposomes 15 min prior to incubation on mica. All samples were rinsed two times with ~ 100 μl volumes of imaging buffer prior to recording images, as described previously [31 (link),32 (link)]. AFM images were acquired in imaging buffer, in tapping mode, using the Cypher or Cypher VRS (Asylum Research, Santa Barbara, CA) and tips (BL-AC40TS, Olympus, Tokyo Japan, or USC-F0.3-k0.3, Nanoworld, Neuchatel Switzerland). Images were acquired at ~ 32 °C with an estimated tip-sample force < 100 pN.
Bl ac40ts
Manufactured by Olympus
Sourced in Japan
The BL-AC40TS is a compact and versatile laboratory centrifuge designed for a variety of applications. It features a maximum speed of 4,000 RPM and a maximum relative centrifugal force (RCF) of 2,010 × g. The centrifuge accommodates a range of rotor types and tube sizes, making it suitable for general-purpose use in research and clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Icon afm, by Bruker (2 mentions)
Ac240tsa r3, by Oxford Instruments (2 mentions)
Igor pro, by Wavemetrics (2 mentions)
V1 grade, by Ted Pella (1 mentions)
Usc f0 k0, by NanoWorld (1 mentions)
Msv 5200, by Jasco (1 mentions)
Mfp 3d bio j, by Oxford Instruments (1 mentions)
Ds qi1mc, by Nikon (1 mentions)
Bx51wi, by Olympus (1 mentions)
Picoplus 5500, by Keysight (1 mentions)
12 protocols using bl ac40ts
1
Imaging Supported Pgp Proteoliposomes
2
AFM Imaging of Biological Samples
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AFM images were collected using an Asylum Research Cypher equipped with an environmental scanner. Details of imaging parameters are the following:
Imaging in air: NSG 01 DLC (nominal parameters: force constant 5.1 N/m, resonant frequency 150 kHz) and Olympus 240 TS (nominal parameters: force constant 2 N/m, resonant frequency 70 kHz) tips. No main differences were found using these two tip models. AFM fast scan rate was around 1–1.5 Hz with a tip oscillation amplitude below 10 nm.
Imaging in liquid: Olympus BL-AC40TS (nominal parameters: force constant 0.09 N/m, resonant frequency 110 kHz in air) tip. Fast scanning rate between 2 and 4 Hz, tip oscillation amplitude between 2 and 4 nm.
3
Nanoscale Height Measurements Using AFM
4
Characterizing Swellable Gold Nanodot Arrays
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After removal of unbounded surface ligands, PAA gels were sequentially immersed in NaCl aqueous solution with various concentrations to change their volumes. The gold nanodot arrays on the gels with various swelling degrees were observed using a transmission microscope (BX51, Olympus Corp., Japan) and AFM (MFP-3D-BIO-J, Oxford Instruments plc, Britain) in solution. AFM imaging was performed in the AC mode with a silicon nitride cantilever (BL-AC40TS, Olympus Corp., Japan). Extinction spectra of the gold nanodot arrays on PAA hydrogel surfaces were measured using an UV-Vis-NIR microspectrometer (MSV-5200, JASCO Corp., Japan).
5
Nanoscale Height Measurements Using AFM
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AFM experiments for height measurements were performed using soft cantilevers (AC240TSA-R3, Oxford Instrument Co.) with a nominal force constant of 2 N/m and resonance frequencies of 70 kHz. The free-air amplitude of the tip was calibrated with the Asylum Research software and the spring constant was captured by the thermal vibration method. Samples were imaged with a Cypher ES scanner using intermittent tapping (AC-air topography) mode. All nanowires height analyses and statistics were performed using Gwyddion and IGOR Pro software (WaveMetrics, Inc.). To check protein quality for infrared nanospectroscopy, AFM was performed using cantilevers (Arrow-NCR, Nano Worlds) with a nominal force constant of 42 N/m and a resonance frequency of 285 kHz. Samples were imaged with an Icon AFM (Bruker) using intermittent tapping (AC-air topography) mode. Liquid AFM was performed using bio-lever mini tips (BL-AC40TS, Olympus) with resonance frequency of 25 kHz in liquid and nominal force of 0.1 N/m. AFM images were processed using the Gwyddion package.
6
Imaging Membrane Protein Complexes by AFM
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Images were acquired in tapping mode in fluid at ~30 °C using a commercial instrument (Cypher, Asylum Research Inc.). Details for each sample preparation follow. SecYEG and SecYEG/SecA complexes : Proteoliposome stock solutions were diluted to 80 nM protein, 80 μM lipid in recording buffer (10 mM HEPES pH 8.0, 200 mM KAc, 5 mM MgAc2), immediately deposited on a freshly plasma cleaned glass support11 (link) or freshly cleaved mica10 (link),41 (link) and incubated for ~20 minutes, followed by rinsing with recording buffer (~300 μl). Biolever mini tips (BL-AC40TS, Olympus) with measured spring constants ~0.06 N/m were used. Spring constants were determined using the thermal noise method. Bacteriorhodopsin on mica : Following established protocols43 (link), equal volumes of stock solution and recording buffer (10 mM Tris pH ~7.6, 150 mM KCl) were mixed before depositing onto a freshly cleaved mica support. After a 1 hr incubation, the sample was rinsed with 300 μl of recording buffer. SNL (Veeco) tips of measured spring constant ~0.4 N/m were used.
7
Formation and Imaging of Supported Lipid Bilayers
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A micellar solution of 0.5 mg/mL POPC with 10% sterols in DDM-containing buffer (10 mM HEPES, pH 7.5, 200 mM KCl, and 0.06% DDM) was diluted 10 times with the buffer without DDM to form liposomes. The liposome solution was placed on a freshly cleaved mica surface. The bilayers were absorbed on the mica surface, resulting in partially formed supported lipid bilayers. After a 15-min incubation, the floating liposomes and detergent were rinsed off with buffer (10 mM HEPES, pH 7.5 and 300 mM KCl). The solution was then replaced with recording solution containing AM3 (10 mM HEPES, pH 7.5, 300 mM KCl, and 4.5 μM AM3). Images were taken with a Cypher (Oxford Instruments) using AC mode at 28 °C. BL-AC40TS (OLYMPUS) cantilevers were used and had a tip radius and spring constant of approximately 7 nm and 0.1 N/m, respectively. Image analysis was performed using Gwyddion software (Department of Nanometrology, Czech Metrology Institute, Czech).
8
Atomic Force Microscopy of Protein Dynamics
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AFM images were acquired at ~32°C in tapping mode using a commercial instrument (Asylum Research Cypher) in 10 mM Hepes (pH 7.6), 100 mM KAc, and 5 mM MgAc2 or 10 mM Hepes (pH 7.6), 100 mM KAc, 5 mM MgAc2 ,and 100 μM nucleotide, as specified. Measurement-related bias of protein conformations and conformational dynamics is an ever present concern. Care was taken to control the magnitude of the tip sample force to ⪝100 pN [estimated by comparing the free amplitude (~10 nm) to the set point amplitude]. BioLever mini tips (BL-AC40TS, Olympus) with measured spring constants of ~0.06 N/m were used. Spring constants were determined using the thermal noise method. AFM images were acquired with the following parameters: scan rate, 3.6 Hz; scan size, 512 pixels × 512 pixels, 1 μm2 × 1 μm2; scan speed, 7 nm/ms. Kymographs were acquired as follows: scan rate, 5.2 Hz; scan size, 320 pixels, 300 nm; scan speed, 3 nm/ms. We analyzed at least 17 images per condition studied, at least 3 independent experiments/mica stages/sample preparations per condition, and at least 2 different tips for each independent experiment. Note that tips were recovered after each experiment and cleaned for reuse.
9
Atomic Force Microscopy of Nanoparticles
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Samples were deposited on poly-ornithine functionalized mica. For the deposition 20 µL of particles solution were left on the mica surface for one hour and then gently rinsed with PBS. The samples were not dried and a drop of PBS solution was always kept on the surface. The measurements were carried out in PBS with an MFP-3D Asylum research instrument in dynamic mode using soft cantilevers (BLAC40- TS Olympus, radius of curvature < 10 nm, spring constant 0.1 N/m). Images were acquired with 8 nm pixel size at 0.75 Hz scan rate in at least 5 different positions on the sample.
10
Characterizing Lipid Bilayer Domains by AFM and Fluorescence
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The SLB samples were observed with an AFM (PicoPlus 5500, Keysight Technologies, Inc., Santa Rosa, USA, formerly Molecular Imaging, Corp.) and an epi-fluorescence microscope (BX51WI, Olympus, Tokyo, Japan) in the buffer solution at 25 °C unless otherwise noted. AFM topographies were obtained in the acoustic AC mode (tapping mode) using a Si3N4 cantilever with a spring constant of 0.1 N/m (BL-AC40TS, Olympus). We obtained AFM topographies at least five different positions of a sample to calculate average area fractions of domains and to confirm the generality of the observed phenomena. Fluorescence images were obtained using a high-pressure mercury lump, a mirror unit (U-MWIG3, with a long-pass emission filter >575 nm) and neutral density (ND) filters and recorded with a CCD camera (DS-Qi1Mc, Nikon, Tokyo, Japan). Pixel size was 0.22 µm/px with a 60× water-immersion lens (LUMPlan FL 60×, NA = 1.00), and the spatial resolution at the wavelength of 580 nm was approximately 350 nm based on the Rayleigh criterion. Area fractions of the domains in the fluorescence images and AFM topographies were obtained with Image J (NIH, USA, https://imagej.nih.gov/ij/ ) and SPIP (Image Metrology A/S, Hørsholm, Denmark), respectively.
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