{"title":"Negative Staining Electron Microscopy of a Highly Flexible Sec1/Munc18 Protein Complex Stabilized by Glutaraldehyde Crosslinking.","authors":"Richard J Y Liu, Walter H A Kahr","doi":"10.1007/978-1-0716-4314-3_16","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"227-235"},"PeriodicalIF":0.0000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in molecular biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/978-1-0716-4314-3_16","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
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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.
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For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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