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

Richard J Y Liu; Walter H A Kahr

doi:10.1007/978-1-0716-4314-3_16

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

Methods Mol Biol. 2025:2887:227-235. doi: 10.1007/978-1-0716-4314-3_16.

Authors

Richard J Y Liu¹, Walter H A Kahr^{2

3

4}

Affiliations

¹ Departments of Paediatrics and Biochemistry, University of Toronto, Toronto, ON, Canada.
² Department of Biochemistry, University of Toronto, Toronto, ON, Canada. walter.kahr@sickkids.ca.
³ Department of Paediatrics, University of Toronto, Toronto, ON, Canada. walter.kahr@sickkids.ca.
⁴ Cell Biology Program, Research Institute and Division of Haematology/Oncology, Hospital for Sick Children, Toronto, ON, Canada. walter.kahr@sickkids.ca.

PMID: 39806158
DOI: 10.1007/978-1-0716-4314-3_16

Abstract

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.

Keywords: Avidin; Crosslinking; Electron microscopy; Negative staining; Protein complex; Sec1/Munc18; Structure determination.

MeSH terms

Biotinylation
Cross-Linking Reagents* / chemistry
Glutaral* / chemistry
Microscopy, Electron* / methods
Multiprotein Complexes / chemistry
Multiprotein Complexes / metabolism
Multiprotein Complexes / ultrastructure
Negative Staining* / methods

Substances

Glutaral
Cross-Linking Reagents
Multiprotein Complexes