The extracted chromatin samples with a concentration of 220 ng/μl were used for freezing. 10 μl of chromatin sample was mixed with 12 μl of 10 nm gold nanoparticle solutions. An aliquot of 3 μl of the mixture was adsorbed onto the glow-discharged 400 mesh Lacey carbon holey grids (Ted Pella, INC, CA, USA) for 1 min, and then manually blotted by putting a piece of #1 filter paper at the edge of the grid at 50% humidity, followed by plunging into liquid ethane cooled by liquid nitrogen inside an FEI Vitrobot Mark III (FEI, Eindhoven).
Lacey holey carbon grids
Manufactured by Ted Pella
Sourced in United States
Lacey holey carbon grids are a type of sample support used in transmission electron microscopy (TEM). They consist of a thin, perforated carbon film supported on a metal grid. The holes in the carbon film allow the electron beam to pass through the sample, enabling high-resolution imaging of the specimen.
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Lab products found in correlation
Vitrobot mark 3, by Thermo Fisher Scientific (5 mentions)
Tecnai g2 twin tem, by Thermo Fisher Scientific (4 mentions)
Easiglow cleaning system, by Ted Pella (3 mentions)
626 cryo holder, by Ametek (3 mentions)
Gatan 626 cryo holder, by Ametek (2 mentions)
Fei vitrobot mark 3, by Thermo Fisher Scientific (1 mentions)
Fei tecnai g2 twin tem, by Thermo Fisher Scientific (1 mentions)
Pelco easiglow apparatus, by Ted Pella (1 mentions)
Pelco easiglow cleaning system, by Ted Pella (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Cary 300, by Agilent Technologies (1 mentions)
Dynapro, by Wyatt Technology (1 mentions)
8 protocols using lacey holey carbon grids
1
Chromatin Freezing and Cryo-EM Sample Preparation
2
Cryo-EM Sample Preparation via Chromatin Adsorption
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The isolated native chromatin samples with a concentration of 220 ng μL−1 were used for CryoEM grid preparation. A mixture solution of native chromatin and gold nanoparticles was added onto glow-discharged 400 mesh Lacey carbon holey grids (Ted Pella, INC, CA, USA) for 1 min, and then manually blotted using a piece of #1 filter paper at the edge of the grid. The grids were allowed to stand for 30–60 s before plunging into liquid ethane cooled by liquid nitrogen inside a FEI Vitrobot Mark III (FEI, Eindhoven). It should be noted that the evaporation time for Cryo-EM sample preparation is sensitive to atmospheric humidity and the preparation time should be experimentally determined.
3
Vitrification of Samples for Cryo-TEM Imaging
4
Cryogenic TEM Imaging of Peptide Assemblies
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Solutions containing 100 μM
E40S–S10Q or E80S–S10Q and incubated
for 16 h at room temperature were prepared
for cryo-TEM. The cryo-TEM measurements were performed at Duke University’s
Shared Materials Instrumentation Facility. First, lacey holey carbon
grids (Ted Pella) were cleaned in a PELCO EasiGlow cleaning system
(Ted Pella). For each sample, 3 μL was deposited onto a grid.
Samples were then vitrified using a FEI Vitrobot Mark III by blotting
the sample for 3 s with an offset of −3 mm and vitrifying it
in liquid ethane. To prevent sample evaporation prior to vitrification,
the sample chamber was kept at 22 °C and 100% humidity. Finally,
the prepared grids were placed into a Gatan 626 cryoholder and imaged
on a FEI Tecnai G2 Twin.
E40S–S10Q or E80S–S10Q and incubated
for 16 h at room temperature were prepared
for cryo-TEM. The cryo-TEM measurements were performed at Duke University’s
Shared Materials Instrumentation Facility. First, lacey holey carbon
grids (Ted Pella) were cleaned in a PELCO EasiGlow cleaning system
(Ted Pella). For each sample, 3 μL was deposited onto a grid.
Samples were then vitrified using a FEI Vitrobot Mark III by blotting
the sample for 3 s with an offset of −3 mm and vitrifying it
in liquid ethane. To prevent sample evaporation prior to vitrification,
the sample chamber was kept at 22 °C and 100% humidity. Finally,
the prepared grids were placed into a Gatan 626 cryoholder and imaged
on a FEI Tecnai G2 Twin.
5
Cryo-TEM imaging of vitrified samples
6
Vitrification of Samples for Cryo-TEM Imaging
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Cryogenic transmission electron microscopy (cryo-TEM) was performed at Duke University’s Shared Materials Instrumentation Facility (Durham, NC). Lacey holey carbon grids (Ted Pella, Redding, CA) were glow discharged in a PELCO EasiGlow Cleaning System (Ted Pella, Redding, CA). A 3 µl drop of a sample was deposited onto the grid, blotted for 3 seconds with an offset of −3 mm, and vitrified in liquid ethane using the Vitrobot Mark III (FEI, Eindhoven, Netherlands). Prior to vitrification, the sample chamber was maintained at 22 °C and 100% relative humidity to prevent sample evaporation. Grids were transferred to a Gatan 626 cryoholder (Gatan, Pleasanton, CA) and imaged on a FEI Tecnai G2 Twin TEM (FEI, Eindhoven, Netherlands).
7
Cryo-TEM Imaging of Elastin-Like Polypeptides
8
Temperature-Dependent Self-Assembly of IDPPs
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Temperature-dependent self-assembly of diblock IDPPs was studied at a fixed concentration of 50 μM in PBS, by optical turbidity measurements on a UV-visible spectrophotometer (Cary 300) and dynamic light scattering measurements on a Wyatt DynaPro temperature-controlled microsampler. Heating and cooling were performed at ~1°C/min. We also conducted Cryo-TEM at Duke University’s Shared Materials Instrumentation Facility (Durham, NC) using Lacey holey carbon grids (Ted Pella, Redding, CA) and a Vitrobot Mark IV (FEI, Eindhoven, The Netherlands) for blotting and vitrification. We triggered nanoparticle assembly by incubating samples (50 μM in PBS) at 50°C for 15 min. We then loaded samples onto grids within the vitrification chamber set to either 50°C (Fig. 6D ) or 30°C (Fig. 6E ) and at 100% relative humidity. After vitrification, grids were transferred to a Gatan 626 cryoholder (Gatan, Pleasanton, CA) and imaged on a FEI Tecnai G2 Twin TEM (FEI, Eindhoven, The Netherlands), operating under low-voltage conditions at 80 keV.
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