Analysis of Normal and Neoplastic Tissue NHC Proteins by High-Resolution Two-Dimensional Gradient Electrophoresis and Silver Staining

Author(s):  
Lewis V. Rodriguez
1982 ◽  
Vol 28 (4) ◽  
pp. 766-781 ◽  
Author(s):  
R Bravo ◽  
J E Celis

Abstract A total of 1357 polypeptides [946 acidic (isoelectric focusing) and 411 basic (nonequilibrium pH-gradient electrophoresis)] from human HeLa cells have been separated and catalogued with use of high-resolution two-dimensional gel electrophoresis. Of these polypeptides, 1266 were detected by labeling cells with [35S]methionine, while the rest were revealed by silver staining or by labeling with a mixture of 16 14C-labeled amino acids. For convenience, all these polypeptides have been numbered and are indicated in a large fold-out protein map. The percentages of some of the major 14C-labeled proteins have been determined, and for some we list a few characteristics such as: variation during the cell cycle; cellular distribution in cytoplasts and karyoplasts; presence in Triton- and salt-extracted cytoskeletons; and phosphorylation and sensitivity to neoplastic transformation.


Author(s):  
H.A. Cohen ◽  
T.W. Jeng ◽  
W. Chiu

This tutorial will discuss the methodology of low dose electron diffraction and imaging of crystalline biological objects, the problems of data interpretation for two-dimensional projected density maps of glucose embedded protein crystals, the factors to be considered in combining tilt data from three-dimensional crystals, and finally, the prospects of achieving a high resolution three-dimensional density map of a biological crystal. This methodology will be illustrated using two proteins under investigation in our laboratory, the T4 DNA helix destabilizing protein gp32*I and the crotoxin complex crystal.


Author(s):  
K. H. Downing ◽  
S. G. Wolf ◽  
E. Nogales

Microtubules are involved in a host of critical cell activities, many of which involve transport of organelles through the cell. Different sets of microtubules appear to form during the cell cycle for different functions. Knowledge of the structure of tubulin will be necessary in order to understand the various functional mechanisms of microtubule assemble, disassembly, and interaction with other molecules, but tubulin has so far resisted crystallization for x-ray diffraction studies. Fortuitously, in the presence of zinc ions, tubulin also forms two-dimensional, crystalline sheets that are ideally suited for study by electron microscopy. We have refined procedures for forming the sheets and preparing them for EM, and have been able to obtain high-resolution structural data that sheds light on the formation and stabilization of microtubules, and even the interaction with a therapeutic drug.Tubulin sheets had been extensively studied in negative stain, demonstrating that the same protofilament structure was formed in the sheets and microtubules. For high resolution studies, we have found that the sheets embedded in either glucose or tannin diffract to around 3 Å.


2001 ◽  
Vol 120 (5) ◽  
pp. A226-A226 ◽  
Author(s):  
W LAMMERS ◽  
S DHANASEKARAN ◽  
J SLACK ◽  
B STEPHEN

1985 ◽  
Vol 54 (03) ◽  
pp. 626-629 ◽  
Author(s):  
M Meyer ◽  
F H Herrmann

SummaryThe platelet proteins of 9 thrombasthenic patients from 7 families were analysed by high resolution two-dimensional gel electrophoresis (HR-2DE) and crossed immunoelectrophoresis (CIE). In 7 patients both glycoproteins (GPs) IIb and Ilia were absent or reduced to roughly the same extent. In two related patients only a trace of GP Ilb-IIIa complex was detected in CIE, but HR-2DE revealed a glycopeptide in the position of GP Ilia in an amount comparable to type II thrombasthenia. This GP Ilia-like component was neither recognized normally by anti-GP Ilb-IIIa antibodies nor labeled by surface iodination. In unreduced-reduced two-dimensional gel electrophoresis two components were observed in the region of GP Ilia. The assumption of a structural variant of GP Ilia in the two related patients is discussed.


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