577 research outputs found
Silver staining of proteins in polyacrylamide gels
Silver staining is used to detect proteins after electrophoretic separation
on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram
range) with the use of very simple and cheap equipment and chemicals. It is
compatible with downstream processing, such as mass spectrometry analysis after
protein digestion. The sequential phases of silver staining are protein
fixation, then sensitization, then silver impregnation and finally image
development. Several variants of silver staining are described here, which can
be completed in a time range from 2 h to 1 d after the end of the
electrophoretic separation. Once completed, the stain is stable for several
weeks
Gene induction during differentiation of human monocytes into dendritic cells: an integrated study at the RNA and protein levels
Changes in gene expression occurring during differentiation of human
monocytes into dendritic cells were studied at the RNA and protein levels.
These studies showed the induction of several gene classes corresponding to
various biological functions. These functions encompass antigen processing and
presentation, cytoskeleton, cell signalling and signal transduction, but also
an increase in mitochondrial function and in the protein synthesis machinery,
including some, but not all, chaperones. These changes put in perspective the
events occurring during this differentiation process. On a more technical
point, it appears that the studies carried out at the RNA and protein levels
are highly complementary.Comment: website publisher:
http://www.springerlink.com/content/ha0d2c351qhjhjdm
Effects of nanoparticles on murine macrophages
Metallic nanoparticles are more and more widely used in an increasing number
of applications. Consequently, they are more and more present in the
environment, and the risk that they may represent for human health must be
evaluated. This requires to increase our knowledge of the cellular responses to
nanoparticles. In this context, macrophages appear as an attractive system.
They play a major role in eliminating foreign matter, e.g. pathogens or
infectious agents, by phagocytosis and inflammatory responses, and are thus
highly likely to react to nanoparticles. We have decided to study their
responses to nanoparticles by a combination of classical and wide-scope
approaches such as proteomics. The long term goal of this study is the better
understanding of the responses of macrophages to nanoparticles, and thus to
help to assess their possible impact on human health. We chose as a model
system bone marrow-derived macrophages and studied the effect of commonly used
nanoparticles such as TiO2 and Cu. Classical responses of macrophage were
characterized and proteomic approaches based on 2D gels of whole cell extracts
were used. Preliminary proteomic data resulting from whole cell extracts showed
different effects for TiO2-NPs and Cu-NPs. Modifications of the expression of
several proteins involved in different pathways such as, for example, signal
transduction, endosome-lysosome pathway, Krebs cycle, oxidative stress response
have been underscored. These first results validate our proteomics approach and
open a new wide field of investigation for NPs impact on macrophagesComment: Nanosafe2010: International Conference on Safe Production and Use of
Nanomaterials 16-18 November 2010, Grenoble, France, Grenoble : France (2010
Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis
Mitochondria are complex organelles, and their proteomics analysis requires a
combination of techniques. The emphasis in this chapter is made first on
mitochondria preparation from cultured mammalian cells, then on the separation
of the mitochondrial proteins with two-dimensional electrophoresis (2DE),
showing some adjustment over the classical techniques to improve resolution of
the mitochondrial proteins. This covers both the protein solubilization, the
electrophoretic part per se, and the protein detection on the gels, which makes
the interface with the protein identification part relying on mass
spectrometry
Detergents and chaotropes for protein solubilization before two-dimensional electrophoresis
Because of the outstanding ability of two-dimensional electrophoresis to
separate complex mixtures of intact proteins, it would be advantageous to apply
it to all types of proteins, including hydrophobic and membrane proteins.
Unfortunately, poor solubility hampers the analysis of these molecules. As
these problems arise mainly in the extraction and isoelectric focusing steps,
the solution is to improve protein solubility under the conditions prevailing
during isoelectric focusing. This chapter describes the use of chaotropes and
novel detergents to enhance protein solubility during sample extraction and
isoelectric focussing, and discusses the contribution of these compounds to
improving proteomic analysis of membrane proteins
Proteomic analysis of the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease
So far, only the detection of 14-3-3 proteins in cerebrospinal fluid (CSF) has been accepted as diagnostic criterion for Creutzfeldt-Jakob disease (CJD). However, this assay cannot be used for screening because of the high rate of false-positive results, whereas patients with variant CJD are often negative for 14-3-3 proteins. The aim of this study was to compare the spot patterns of CSF by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to search for a CJD-specific spot pattern. We analyzed the CSF of 28 patients {[}11 CJD, 9 Alzheimer's disease ( AD), 8 nondemented controls (NDC)] employing 2D-PAGE which was optimized for minimal volumes of CSF (0.1 ml; 7-cm strips). All samples were run at least three times, gels were silver stained and analyzed by an analysis software and manually revised. We could consistently match 268 spots which were then compared between all groups. By the use of 5 spots, we were able to differentiate CJD from AD or NDC with a sensitivity of 100%. CJD could also be distinguished from both groups by using a heuristic clustering algorithm of 2 spots. We conclude that this proteomic approach can differentiate CJD from other diseases and may serve as a model for other neurodegenerative diseases. Copyright (C) 2007 S. Karger AG, Basel
Solubilization of Proteins in 2DE: An Outline
Protein solubilization for two-dimensional electrophoresis (2DE) has to break
molecular interactions to separate the biological contents of the material of
interest into isolated and intact polypeptides. This must be carried out in
conditions compatible with the first dimension of 2DE, namely isoelectric
focusing. In addition, the extraction process must enable easy removal of any
nonprotein component interfering with the isoelectric focusing. The constraints
brought in this process by the peculiar features of isoelectric focusing are
discussed, as well as their consequences in terms of possible solutions and
limits for the solubilization process
The Whereabouts of 2D Gels in Quantitative Proteomics
Two-dimensional gel electrophoresis has been instrumental in the development
of proteomics. Although it is no longer the exclusive scheme used for
proteomics, its unique features make it a still highly valuable tool,
especially when multiple quantitative comparisons of samples must be made, and
even for large samples series. However, quantitative proteomics using 2D gels
is critically dependent on the performances of the protein detection methods
used after the electrophoretic separations. This chapter therefore examines
critically the various detection methods (radioactivity, dyes, fluorescence,
and silver) as well as the data analysis issues that must be taken into account
when quantitative comparative analysis of 2D gels is performed
Epidemiology of paraneoplastic neurologic syndromes and autoimmune encephalitides in France
OBJECTIVE: To determine the observed and expected incidence rates of paraneoplastic neurologic syndromes (PNSs) and autoimmune encephalitides (AEs) diagnosed in France between 2016 and 2018, we conducted a population-based epidemiologic study. METHODS: Observed incidence rates were stratified by sex, age groups, region of care, year of diagnosis, and disease subgroups. National expected incidence rates were calculated based on rates obtained in the area directly adjacent to the Reference Center using a mixed Poisson model and compared with observed incidence rates. RESULTS: Six hundred thirty-two patients with definite PNS or AE met the inclusion criteria. The observed incidence rate of definite PNS and AE in France was 3.2 per million person-years (CI95%: 2.9-3.4) compared with an expected incidence rate of 7.1 per million person-years (CI95%: 3.9-11.4). The national observed incidence rate for the antibody-positive AE subgroup increased from 1.4 per million person-years (CI95%: 1.2-1.7) in 2016 to 2.1 per million person-years (CI95%: 1.7-2.4) in 2018, thus surpassing the incidence rate of classical PNS (1.2 per million person-years [CI95%: 1.0-1.5]) of 2018. CONCLUSIONS: There was a significant widespread year-to-year increase in the incidence of diagnoses registered with the Reference Center for all subgroups of PNS and AE studied. The national observed incidence rate is likely underestimated due to underdiagnosis and underreporting
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