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Technology Topics Reagents

Just blot and see

SBKB [doi:10.1038/sbkb.2011.69]
Technical Highlight - March 2012
Short description: A simple method for transferring proteins onto EM grids may speed up single-particle analyses of large protein complexes.

Single-particle analysis uses large numbers of transmission electron micrographs to produce a relatively high-resolution electron density map of macromolecular assemblies. It has certain advantages over crystallographic or NMR structural determination techniques: it requires relatively little protein sample (5–10 ng of protein sample is sufficient to cover an electron microscopy (EM) grid) and it can tolerate some sample heterogeneity, as computational algorithms used to process and classify the micrograph images can eliminate unwanted sample populations.

Negative-stain micrographs of thermosomes (left), 20 S proteasomes (center) and VCP-like ATPase (right) obtained by grid blotting.1

Protein purification is a limitation to single-particle analysis. It is very often tailored to the specific complex and can be time consuming, creating a bottleneck for high-throughput workflows. Baumeister and colleagues have developed a simple method for protein complex purification and preparation for EM called 'grid blotting'. The technique uses native gel electrophoresis to focus the protein sample of interest into a sharp band — ideally, the sample could be isolated in this fashion after crude cell lysate fractionation, but preliminary purification steps may be needed. The protein band of interest would then be transferred in a blotting step directly to the EM grid for standard sample preparation and image collection.

The grid-blotting procedure was tested on three complexes from Thermoplasma acidophilum — the thermosome (940 kD), 20S proteasome (680 kD) and VCP-like ATPase(500 kD) — after purification from the crude cell lysate cytosolic fraction using two ion-exchange chromatography steps. Transfer efficiencies were better for negatively stained samples, which use a 10 nm-thick carbon-coated copper grid, than for unstained, vitrified samples, which use a much thinner grid.

The average images obtained from this method were consistent with the known structures of the complexes examined, and there seems to be no need to adjust the method to the size of the complex being examined. Size limitations are dictated by the size range of proteins able to be examined by EM (> 200 kDa) and the native gel matrix (complexes > 4 MDa cannot enter the gel matrix). The method is simple enough to be used by those not familiar with protein purification and can be incorporated into a high-throughput pipeline, as there is sufficient protein isolated for EM and mass spectrometric analysis.

Michelle Montoya

References

  1. R.W. Knispel et al. Blotting protein complexes from native gels to electron microscopy grids. Nat. Meth. 9, 182-184 (2012).
    doi:10.1038/nmeth.1840

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