Negative staining of proteins

N. A. Kiselev, Michael Sherman, V. L. Tsuprun

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Negative staining, some closely related alternative preparation techniques and radiation stability are considered. An attempt is made to clarify the mechanism of action and ultimate resolution limit of negative staining. The results of electron diffraction investigation of thermitase micocrystals embedded in glucose and glucose + stains are presented. It is shown that at doses not exceeding 10 electrons/nm2 electron diffraction from thermitase crystals demonstrate diffraction fields up to 0.2 nm. When adding heavy-atom salts to glucose or using negative staining, the relative intensities of reflections change and electron diffraction patterns for every type of heavy-atom additive (or negative stain) have their specific features. Such characteristic changes of reflection intensities indicate specific interaction of these additives (or stains) with the object. In the case of electron diffraction from the crystals stained using the routine negative staining technique the ordering was preserved down to 0.4-0.5 nm. Increasing the dose up to the normal value results in fading of distant reflections. Thus, negative staining with radiation doses less than the critical one could yield resolution down to 0.4 nm. Yet, the structure may change due to interaction with the stain. Nevertheless, the possibility that such resolution could be obtained for a limited number of objects should not be excluded. Some examples of the application of negative staining for investigation of quaternary and domain structure of proteins (nitrogenase, glutamine synthetase, mitochondrial ATP-synthase, membrane monooxygenase enzymes), tubular and two-dimensional protein crystals (catalase, phosphorylase, HWV protein, hydrogenase), as well as ribosomes and bacteriophages are given in the review.

Original languageEnglish (US)
Pages (from-to)43-72
Number of pages30
JournalElectron Microscopy Reviews
Volume3
Issue number1
DOIs
StatePublished - 1990
Externally publishedYes

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Negative Staining
Electrons
Coloring Agents
Proteins
Glucose
Quaternary Protein Structure
Mitochondrial Proton-Translocating ATPases
Radiation
Hydrogenase
Nitrogenase
Phosphorylases
Glutamate-Ammonia Ligase
Mixed Function Oxygenases
Ribosomes
Bacteriophages
Catalase
Reference Values
Salts
Membranes
Enzymes

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Negative staining of proteins. / Kiselev, N. A.; Sherman, Michael; Tsuprun, V. L.

In: Electron Microscopy Reviews, Vol. 3, No. 1, 1990, p. 43-72.

Research output: Contribution to journalArticle

Kiselev, N. A. ; Sherman, Michael ; Tsuprun, V. L. / Negative staining of proteins. In: Electron Microscopy Reviews. 1990 ; Vol. 3, No. 1. pp. 43-72.
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