Home › Microscopy & Imaging › Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Microscopy & Imaging JoVE (Open Access) Citable · DOI

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

DOI: 10.3791/51087-v
What you'll learn
  • ✓ Prepare samples for negative staining electron microscopy imaging
  • ✓ Execute optimized negative staining protocol for small proteins
  • ✓ Analyze EM grids and interpret high-resolution protein structure images
  • ✓ Evaluate image quality for three-dimensional reconstruction
Protocol

More than half of proteins are small proteins (molecular mass <200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Difficulty
advanced
Total time
~2-4 hours per sample batch (including grid preparation, staining, and imaging)

Steps

1
Prepare materials and equipment for staining

Gather and organize all required materials, reagents, and electron microscopy equipment needed for the negative staining protocol. Ensure all components are ready before beginning sample preparation.

▶ 01:56
2
Apply optimized negative staining to protein samples

Execute the optimized negative staining protocol on prepared protein samples to achieve high contrast and ~1 nm resolution imaging suitable for small asymmetric proteins or complexes.

▶ 03:42
3
Examine and assess EM grid quality

Load stained grids into the electron microscope and evaluate image quality, contrast, and protein distribution across the sample for suitability in three-dimensional reconstruction.

▶ 06:16
4
Interpret representative optimized negative staining results

Review example images and datasets demonstrating successful optimized negative staining outcomes for various small and asymmetric protein structures under different physiological conditions.

▶ 07:18
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