Negative staining is a simple sample preparation method in which protein samples are adsorbed to a continuous carbon film and embedded in a thin layer of dried heavy metal salt to increase specimen contrast. The enhanced contrast of negative stain EM allows viewing of relatively small biological samples (>100kDa).<\/p>\n
This method is used mainly to determined 3D structure of small protein and protein complex (<200kDa), assess sample quality (e.g. purity and homogeneity) and determined initial 3D structure using the Random Conical Tilt (RCT) method. It can also be used to answer specific questions concerning protein-protein interactions and identify protein\/domain assembly. Antibodies or Fab can be visualized when bound to their antigen.<\/p>\n
<\/p>\n
Benefits and Drawbacks of Negative Stain<\/strong><\/p>\n Benefits:<\/strong><\/em><\/p>\n Drawbacks:<\/strong><\/em><\/p>\n Protocols for Negative Staining<\/a><\/p>\n Further reading:<\/strong><\/p>\n “Negative Staining and Image Classification – Powerful Tools in Modern Electron Microscopy<\/a>” – M. Ohi, Y. Li, Y. Cheng, T. Walz (2004) Cryo TEM is used to obtain high resolution 3D structures of protein complexes.<\/p>\n Sample (1-3 \u03bcl) is applied to a holey carbon grid and plunge-frozen in a layer of vitreous ice that preserves the native condition of the protein. Sample images are recorded at cryogenic temperatures using a direct electron detector (K2 Summit on our Polara microscope) or CCD (on our F20 microscope). The low contrast of this method usually necessitates the size of the biological sample to be above ~200kDa.<\/p>\n Direct electron detectors increase the image contrast and when operated in dose fractionation mode (recording a series of low dose images) allows for drift correction.<\/p>\n Data collection is semi-automated by using SerialEM. The raw micrographs are pre-processed on-the-fly by a GPU computer to examine the quality of the data.<\/p>\n","protected":false},"excerpt":{"rendered":" Negative Stain TEM & Cryo TEM Negative Stain Transmission Electron Microscopy (TEM) Negative staining is a simple sample preparation method in which protein samples are adsorbed to a continuous carbon film and embedded in a thin layer of dried heavy metal salt to increase specimen contrast. The enhanced contrast of negative stain EM allows viewing…<\/p>\n","protected":false},"author":1185,"featured_media":0,"parent":1166,"menu_order":1,"comment_status":"closed","ping_status":"closed","template":"","meta":{"spay_email":""},"tags":[],"acf":[],"jetpack_sharing_enabled":true,"_links":{"self":[{"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/pages\/1176"}],"collection":[{"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/users\/1185"}],"replies":[{"embeddable":true,"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/comments?post=1176"}],"version-history":[{"count":21,"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/pages\/1176\/revisions"}],"predecessor-version":[{"id":1248,"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/pages\/1176\/revisions\/1248"}],"up":[{"embeddable":true,"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/pages\/1166"}],"wp:attachment":[{"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/media?parent=1176"}],"wp:term":[{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.vanderbilt.edu\/csb\/wp-json\/wp\/v2\/tags?post=1176"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}\n
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\n“EM Analysis of Protein Structure<\/a>” – M. Ohi (2009)<\/p>\n
\nCryo TEM<\/h4>\n