High-Throughput Protein Analysis Using Negative Stain Electron Microscopy and 2D Classification

2019 ◽  
pp. 477-485
Author(s):  
Christopher P. Arthur ◽  
Claudio Ciferri
Keyword(s):  
Electron Microscopy ◽  
High Throughput ◽  
Protein Analysis ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy

scholarly journals Temperature-dependent reversible assembly of taxol-treated microtubules.

1987 ◽  
Vol 105 (6) ◽  
pp. 2847-2854 ◽  
Author(s):  
C A Collins ◽  
R B Vallee
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
Temperature Dependent ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Microtubule Associated Proteins ◽  
Negative Stain ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Negative Stain Electron Microscopy

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.

scholarly journals Isolation of the yeast nuclear pore complex.

1993 ◽  
Vol 123 (4) ◽  
pp. 771-783 ◽  
Author(s):  
M P Rout ◽  
G Blobel
Keyword(s):  
Electron Microscopy ◽  
Image Reconstruction ◽  
Nuclear Pore Complex ◽  
Sedimentation Coefficient ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy ◽  
Pore Complexes

Nuclear pore complexes (NPCs) have been isolated from the yeast Saccharomyces. Negative stain electron microscopy of the isolated NPCs and subsequent image reconstruction revealed the octagonal symmetry and many of the ultrastructural features characteristic of vertebrate NPCs. The overall dimensions of the yeast NPC, both in its isolated form as well as in situ, are smaller than its vertebrate counterpart. However, the diameter of the central structures are similar. The isolated yeast NPC has a sedimentation coefficient of approximately 310 S and an M(r) of approximately 66 MD. It retains all but one of the eight known NPC proteins. In addition it contains as many as 80 uncharacterized proteins that are candidate NPC proteins.

scholarly journals The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy

2015 ◽  
Vol 1848 (2) ◽  
pp. 496-501 ◽  
Author(s):  
Vincent Postis ◽  
Shaun Rawson ◽  
Jennifer K. Mitchell ◽  
Sarah C. Lee ◽  
Rosemary A. Parslow ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Membrane Protein ◽  
Structure Study ◽  
Protein Scaffold ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy

scholarly journals Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

2021 ◽  
Vol 17 (4) ◽  
pp. e1008977
Author(s):  
Rajesh Kumar ◽  
Suprit Deshpande ◽  
Leigh M. Sewall ◽  
Gabriel Ozorowski ◽  
Christopher A. Cottrell ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Neutralizing Antibodies ◽  
Natural Infection ◽  
Neutralizing Antibody ◽  
Viral Envelope ◽  
Negative Stain ◽  
Clade C ◽  
Negative Stain Electron Microscopy ◽  
Env Protein ◽  
Hiv 1

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping.

Optimization of diagnostic EM for enteric viruses

1992 ◽  
Vol 50 (1) ◽  
pp. 678-679
Author(s):  
C. D. Humphrey ◽  
H.H. Huang ◽  
H. Tanaka
Keyword(s):  
Electron Microscopy ◽  
Enteric Viruses ◽  
Virus Identification ◽  
Negative Stain ◽  
Enteric Adenovirus ◽  
Negative Stain Electron Microscopy ◽  
Viral Diagnosis

Virus identification by negative stain electron microscopy (EM) is one of the most versatile and sensitive assays for rapid viral diagnosis. Electron microscopy is particularly important for the study of enteric viruses because they are difficult to cultivate and characterize. The gastroenteritis viruses most frequently identified include rotavirus (Fig. 1), enteric adenovirus (Fig. 2), enteric coronavirus (Fig. 3), calicivirus (Fig. 4), Norwalk agent, and astrovirus (Fig. 6). In addition, there are several unclassified viruses categorized as either small round structured viruses (Fig. 5) or small round viruses (Fig. 7).

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