Blotting protein complexes from native gels to electron microscopy grids

Knispel, Roland Wilhelm; Kofler, Christine; Boicu, Marius; Baumeister, Wolfgang; Nickell, Stephan

doi:10.1038/nmeth.1840

Cited by 11 publications

(10 citation statements)

References 12 publications

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“…Alternatively, transmission electron microscopy (TEM) could be used. TEM imaging of proteins can be implemented by fabricating gold nanostructures on a TEM membrane, attaching proteins, and staining the attached proteins with uranyl acetate …”

Section: Resultsmentioning

confidence: 99%

The Orientations of Large Aspect‐Ratio Coiled‐Coil Proteins Attached to Gold Nanostructures

Chang

Kim

Thompson

et al. 2016

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Methods for patterning biomolecules on a substrate at the single molecule level have been studied as a route to sensors with single-molecular sensitivity or as a way to probe biological phenomena at the single-molecule level. However, the arrangement and orientation of single biomolecules on substrates has been less investigated. Here, the arrangement and orientation of two rod-like coiled-coil proteins, cortexillin and tropomyosin, around patterned gold nanostructures is examined. The high aspect ratio of the coiled coils makes it possible to study their orientations and to pursue a strategy of protein orientation via two-point attachment. The proteins are anchored to the surfaces using thiol groups, and the number of cysteine residues in tropomyosin is varied to test how this variation affects the structure and arrangement of the surface-attached proteins. Molecular dynamics studies are used to interpret the observed positional distributions. Based on initial studies of protein attachment to gold post structures, two 31-nm-long tropomyosin molecules are aligned between the two sidewalls of a trench with a width of 68 nm. Because the approach presented in this study uses one of twenty natural amino acids, this method provides a convenient way to pattern biomolecules on substrates using standard chemistry.

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Section: Resultsmentioning

confidence: 99%

The Orientations of Large Aspect‐Ratio Coiled‐Coil Proteins Attached to Gold Nanostructures

Chang

Kim

Thompson

et al. 2016

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“…These proteins appeared in the molecular-weight range of around 34 kDa on SDS-PAGE. Ta1207 was further separated from the other proteins using gel-blotting technology (Knispel et al, 2012) and was analyzed by cryo-EM.…”

Section: Methodsmentioning

confidence: 99%

“…The interaction between the proteins was weak, if there was any, as the Ta0425-Ta0424 complex eluted at 500 kDa, while Ta1207 eluted at 200 kDa, in SEC experiments. Ta1207 was further separated from contaminating proteins by native PAGE, transferred to EM grids using gel-blotting technology (Knispel et al, 2012) and analyzed by cryo-EM. Numerous, fairly uniform particles of $110 Å diameter were readily detectable in the sample (Fig.…”

Section: Ta1207mentioning

confidence: 99%

Crystal structure of theThermoplasma acidophilumprotein Ta1207

Pathare

Nagy

Hubert

et al. 2017

Acta Crystallogr F Struct Biol Commun

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The crystal structure of the Ta1207 protein from Thermoplasma acidophilum is reported. Ta1207 was identified in a screen for high-molecular-weight protein complexes in T. acidophilum. In solution, Ta1207 forms homopentamers of 188 kDa. The crystal structure of recombinant Ta1207 solved by Se-MAD at 2.4 Å resolution revealed a complex with fivefold symmetry. In the crystal lattice, calcium ions induce the formation of a nanocage from two pentamers. The Ta1207 protomers comprise two domains with the same novel α/β topology. A deep pocket with a binding site for a negatively charged group suggests that Ta1207 functions as an intracellular receptor for an unknown ligand. Homologues of Ta1207 occur only in Thermoplasmatales and its function might be related to the extreme lifestyle of these archaea. The thermostable Ta1207 complex might provide a useful fivefold-symmetric scaffold for future nanotechnological applications.

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“…At least for cytosolic proteins it might suffice to lyse a couple of cells, extract the target (endogenous) protein using a microfluidic device, and deposit the extracted particles on an EM grid for analysis. The minimal amount of protein needed for a negative stain analysis has already been demonstrated by the direct transfer of proteins from a native polyacrylamide gel to EM grids …”

Section: New Opportunities and Outlookmentioning

confidence: 99%

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”

et al. 2018

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This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.

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Blotting protein complexes from native gels to electron microscopy grids

Cited by 11 publications

References 12 publications

The Orientations of Large Aspect‐Ratio Coiled‐Coil Proteins Attached to Gold Nanostructures

The Orientations of Large Aspect‐Ratio Coiled‐Coil Proteins Attached to Gold Nanostructures

Crystal structure of theThermoplasma acidophilumprotein Ta1207

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”

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