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Negative staining electron microscopy is one of the easiest ways to determine the shape and dimensions of multimeric protein complexes over 100 kDa molecular weight. This method requires small volumes (< 10 μL) of dilute protein (0.01–0.1 mg/mL). Here we describe a method for quickly crosslinking a protein sample and preparing negative stained grids, and we also describe how to label a biotinylated protein subunit with avidin to determine its position within a complex using negative staining EM. This method should be generally applicable for most soluble protein complexes.

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Negative Staining Electron Microscopy of a Highly Flexible Sec1/Munc18 Protein Complex Stabilized by Glutaraldehyde Crosslinking

  • 实验方案

  • 发表日期：

    2025年

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  • Richard J. Y. Liu,
  • Walter H. A. Kahr

摘要

Negative staining electron microscopy is one of the easiest ways to determine the shape and dimensions of multimeric protein complexes over 100 kDa molecular weight. This method requires small volumes (< 10 μL) of dilute protein (0.01–0.1 mg/mL). Here we describe a method for quickly crosslinking a protein sample and preparing negative stained grids, and we also describe how to label a biotinylated protein subunit with avidin to determine its position within a complex using negative staining EM. This method should be generally applicable for most soluble protein complexes.