Purified Ypk9 in nanodiscs was frozen at a concentration of 0.8–1.2 mg/mL. Quantifoil 1.2/1.3 holy carbon grids were glow-discharged using a Leica Coater ACE 200 for 40 s with 10 mA current. The grids were prepared using a Vitrobot Mark IV operated at 100% humidity and 4 °C. In total, 3 µL of purified protein was applied to each grid, incubated for 5 s, blotted for 3 s, and then plunge frozen into liquid ethane. Frozen grids were stored in liquid nitrogen until data collection. The different protein conformations were stabilized using supplements added immediately before freezing: E2Pinhib (1 mM BeF3−); E2P* (1 mM SPM and 1 mM BeF3−); E2.PiALF/SPM (1 mM SPM and 1 mM AlF); E2.PiSPM (1 mM SPM and 1 mM MgCl2). All SPM containing samples were first incubated for 1 h at 18 °C and then heat-activated in a 40 °C water bath for 5 min immediately before freezing. The E2Pinhib sample was only incubated at 18 °C without heat treatment.
Coater ace 200
Manufactured by Leica
Sourced in Austria
The Coater ACE 200 is a high-performance vacuum coating system designed for the deposition of thin films. It provides a controlled environment for the evaporation and condensation of materials onto substrates, enabling the creation of precise, uniform coatings. The system features a compact design and advanced control capabilities to ensure consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Quanta 3d feg sem, by Thermo Fisher Scientific (2 mentions)
Veleta camera, by Olympus (2 mentions)
Whatman, by Cytiva (2 mentions)
Quanta 3d feg, by Thermo Fisher Scientific (2 mentions)
Whatman filter paper, by Cytiva (1 mentions)
Whatman paper, by Cytiva (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Cpd 030, by Leica (1 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (1 mentions)
Reddot, by Biotium (1 mentions)
Axioplan imager z1m microscope, by Zeiss (1 mentions)
Phenom pro scanning electron microscope, by Thermo Fisher Scientific (1 mentions)
12 protocols using coater ace 200
1
Cryo-EM Structure of Ypk9 Nanodiscs
2
Morphological Analysis of Phospholipid Fibers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The morphology of the electrospun phospholipid, phospholipid/vanillin and phospholipid/curcumin fibers was investigated using a Quanta FEG 3D scanning electron microscope (SEM). Samples were attached on metal stubs with double-sided adhesive carbon tape and coated with 6 nm of gold for better conductivity using a sputter coater (Leica Coater ACE 200). The average fiber diameter was calculated using image J analysis software (National Institutes of Health, MD, USA) measured at 100 different points for each image. Changes in morphology of electrospun phospholipid fibers were further monitored after contact in PBS by Environmental SEM (ESEM). Samples were mounted on aluminium stubs and placed upside down immersed in PBS for 1, 2 and 4 h prior to the visualization in the Quanta FEG 3D SEM.
3
Cryo-EM sample preparation for VAR2CSA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For apo sample preparation, a frozen aliquot of VAR2CSA was subjected to size-exclusion chromatography in 20 mM Tris pH 7.5, 125 mM KCl. The generated protein peak was concentrated to 8.6 mg/ml and fluorinated Fos-Cholin8 (FC8) was added to the protein sample to a final concentration of 1 mM immediately before freezing. Quantifoil 1.2/1.3 holy carbon grids were glow-discharged with a Leica Coater ACE 200 for 30 s using 5 mA current. Cryo-EM grids were prepared with a Vitrobot Mark IV operated at 100% humidity and 4 °C. A 3.5 µl aliquot of purified protein was applied to the grids, incubated for 5 s, blotted 3.5 s and plunge frozen into liquid ethane. The sample with plCS was prepared by incubating VAR2CSA with plCS (purified as previously described33 (link),34 (link)) in a 1:7 mass ratio for 30 min at 18 °C. The sample was chondroitinase ABC treated with 300mU enzyme for 1.5 h and further purified using a Superdex200 increase 10/300GL column equilibrated in 20 mM Tris pH 7.5 and 75 mM KCl. Peak fractions were concentrated to 0.5 mg/ml. The cryo-freezing conditions were identical except no FC8 was added.
4
Scanning Electron Microscopy of Fibers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ch/P fibers were mounted on aluminum stubs and sputter-coated (Leica Coater ACE 200 Brønshøj, Denmark) with a layer of 6 nm of gold prior to visualization in a scanning electron microscope (SEM) (Quanta FEG 3D SEM, FEI, Hillsboro, OR, USA). The average fiber diameters and diameter distributions were determined by measuring 100 fibers using the image visualization software Image–J (National Institutes of Health, Bethesda, MD, USA)
5
Visualizing Bacteriophage AV101 by TEM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To visualize phage AV101 with transmission electron microscopy we used a previously described method (Ackermann 2009 ). Briefly, bacteriophages from high-titer stock were sedimented at 12 000 g for 60 min at 4°C and washed three times with Ammonium Acetate (0.1 M, pH 7). Final sediment was used for imaging. 200 mesh copper coated carbon grids (Ted Pella, Inc.) were made hydrophilic by glow discharging the grids using a Leica Coater ACE 200 for 30 s at 10 mA. A volume of 6 µl of phages at a PFU ml−1 of 1012 were pipetted on the grids and incubated for 30 s. All liquid was removed with a Whatman filter paper. The phages were stained by incubating the grid with 6 µl of 2% uranyl acetate for 30 s. In a washing step, 6 µl of ddH2O was pipetted on the grid, incubated for 30 s, and removed with a Whatman paper. The phages where imaged using a CM100 microscope with a Bio TWIN objective lens and a LaB6 emitter. Pictures were taken with an Olympus Veleta camera and analyzed using ImageJ to determine phage particle measurements.
6
Structural Analysis of VAR2CSA-PAM1.4 Fab Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full-length VAR2CSA was complexed with PAM1.4 Fab by mixing at 1:1.5 molar ratio (VAR2CSA: PAM1.4 Fab) and the final concentration of VAR2CSA was 0.8mg/ml. Quantifoil 1.2/1.3 carbon grids were glow-discharged with Leica Coater ACE200 for 60s at 5mA current. Cryo-EM grids were prepared with Vitrobot Mark IV (Thermo Fisher) operated at 100% humidity and 4°C. 3μl of the complexed sample was applied to the grids, incubated 3s, blotted for 3s and plunge frozen in liquid ethane and then stored in liquid nitrogen for data collection [13 (link)].
7
Phage Morphology Visualization by TEM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Electron microscopy was performed to visualize the morphology of the isolated phages using an aliquot of 5 µL from pure suspensions with glow discharge 200 mesh copper-coated grids (Ted Palla, Inc.) using Leica Coater ACE 200 (30 s, 10 mA). Phages on the grids were incubated for 30 s before bloting off liquid using a Whatman filter paper. Samples were then fixed with 5 µL of glutaraldehyde, incubated for 10 s, and blot off excess liquid. Then, samples were stained with 3 µL of 2% uranyl acetate and incubated for 30 s. The transmission electron microscope used was CM100, with a LaB6 emitter. TEM images were taken with an Olympus Veleta camera and analyzed with ImageJ software.
8
Scanning Electron Microscopy of Caseinate Films
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The surface morphology of treated and control caseinate films was determined following Oh et al. ( 2016) with some modifications. The cross-section microstructure of the fabricated films was visualised by use of a Quanta FEG 3D scanning electron microscope (SEM) at an accelerating voltage of 10 kV. Cryo-fractured pieces of films were attached on SEM specimen by a double-sided carbon adhesive tape followed by sputtering with a layer of gold (Leica Coater ACE 200).
9
Visualizing Biofilm Formation in NG-Tubes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To visualize the presence of a biofilm on the interior surface, we flushed a used NG-tube with 1 ml saline to remove residual liquid and cut 1-cm pieces from the proximal part and the distal (intragastric) part of the tube which we then dissected longitudinally to reveal the inner surface. The specimens were fixed in 2% glutaraldehyde in 0.05 M sodium phosphate buffer of pH 7.4. Following three rinses in 0.15 M sodium phosphate buffer (pH 7.4), specimens were postfixed in 1% OsO4 in 0.12 M sodium cacodylate buffer (pH 7.4) for 2 h. Following a rinse in distilled water, the specimens were dehydrated to 100% ethanol according to standard procedures and critical point dried (CPD 030, Leica, Wetzlar, Germany) employing CO2, and the specimens were subsequently mounted on stubs using colloidal coal as an adhesive, and sputter coated with gold (Leica Coater ACE 200). The inner surface of the NG-tube was then examined with a FEG30 scanning electron microscope (Phillips medical systems, Eindhoven, The Netherlands) operated at an accelerating voltage of 2 kV to visualize the presence of a biofilm.
10
Morphological Analysis of Probiotic Encapsulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The size and morphology of the electrosprayed ETC core–shell capsules and free Bifido were investigated using a scanning electron microscope (SEM). Free Bifido samples were fixed with glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) solution (2.5%, v/v in 0.1 M phosphate-buffered solution, pH 7.4) for 1 h. The samples were then dehydrated in a graded ethanol series (50, 70, 90, 96, and 100%) followed by immersion in hexamethyldisilazane (HMDS). Samples (free Bifido and ETC core–shell capsules loaded with Bifido) were coated with 6 nm of gold for better conductivity using a sputter coater (Leica Coater ACE 200, Leica, Vienna, Austria) and imaged in a Quanta FEG 3D SEM (FEI, Eindhoven, Netherlands). The microcapsule diameter was calculated using Image J analysis software (National Institutes of Health, Bethesda, MD, USA), by measuring 50 different capsule diameters for each sample. The average diameter was calculated from these measurements for each microcapsule.
To evaluate probiotics distribution inside the capsules, the microcapsules with encapsulated Bifido cells were dispersed in NaCl 0.9%. Incubation with RedDot (Biotium, Fremont, CA, USA) at 37 °C for 10 min protected from light was performed prior to visualization using an Axioplan Imager Z1m microscope (Zeiss, Oberkochen, Germany).
To evaluate probiotics distribution inside the capsules, the microcapsules with encapsulated Bifido cells were dispersed in NaCl 0.9%. Incubation with RedDot (Biotium, Fremont, CA, USA) at 37 °C for 10 min protected from light was performed prior to visualization using an Axioplan Imager Z1m microscope (Zeiss, Oberkochen, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!