56 research outputs found
Silver Staining of Proteins in 2DE Gels
Silver staining detects proteins after electrophoretic separation on
polyacrylamide gels. Its main positive features are its excellent sensitivity
(in the low nanogram range) and the use of very simple and cheap equipment and
chemicals. 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 day after the end of the electrophoretic separation.
Once completed, the stain is stable for several weeks
Detergents and Chaotropes for Protein Solubilization before Two-Dimensional Electrophoresis
Because of the outstanding separating capabilities of two-dimensional
electrophoresis for complete proteins, it would be advantageous to be able to
apply it to all types of proteins. Unfortunately, severe solubility problems
hamper the analysis of many classes of proteins, but especially membrane
proteins. These problems arise mainly in the extraction and isoelectric
focusing steps, and solutions are sought to improve protein solubility under
the conditions prevailing during isoelectric focusing. These solutions deal
mainly with chaotropes and new detergents, which are both able to enhance
protein solubility. The input of these compounds in proteomics analysis of
membrane proteins is discussed, as well as future directions.Comment: link to publisher's site http://biomed.humanapress.com
Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myelo\"id cell lines nuclear proteomes
One of the challenges of the proteomic analysis by 2D-gel is to visualize the
low abundance proteins, particularly those localized in organelles. An
additional problem with nuclear proteins lies in their strong interaction with
nuclear acids. Several experimental procedures have been tested to increase, in
the nuclear extract, the ratio of nuclear proteins compared to contaminant
proteins, and also to obtain reproducible conditions compatible with 2D-gel
electrophoresis. The NaCl procedure has been chosen. To test the interest of
this procedure, the nuclear protein expression profiles of macrophages and
dendritic cells have been compared with a proteomic approach by 2D-gel
electrophoresis. Delta 2D software and mass spectrometry analyses have allowed
pointing out some proteins of interest. We have chosen some of them, involved
in transcriptional regulation and/or chromatin structure for further
validations. The immunoblotting experiments have shown that most of observed
changes are due to post-translational modifications, thereby a exemplifying the
interest of the 2D gel approach. Finally, this approach allowed us to reach not
only high abundance nuclear proteins but also lower abundance proteins, such as
the HP1 proteins and reinforces the interest of using 2DE-gel in proteomics
because of its ability to visualize intact proteins with their modifications
Organelle proteomics
This unit describes strategies for studying the proteomes of organelles,
which is one example of targeted proteomics. It relies heavily on previously
published units dealing with organelle preparation, protein solubilization, and
proteomics techniques. A specific commentary for organelle proteomics is
provided. Specific protocols for the isolation of nuclei from various sources
(cell cultures, tissues) are also provided
Organelle proteomics.
International audienceThis unit describes strategies for studying the proteomes of organelles, which is one example of targeted proteomics. It relies heavily on previously published units dealing with organelle preparation, protein solubilization, and proteomics techniques. A specific commentary for organelle proteomics is provided. Specific protocols for the isolation of nuclei from various sources (cell cultures, tissues) are also provided
About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis.
web publisher www.interscience.wiley.comInternational audienceThe influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in-gel rehydration on wide gradients (e.g. 3-10 and 4-12), but anodic cup-loading was still required for basic gradients (e.g. 6-12 or 8-12). These results demonstrate that thiol-related problems are not solely responsible for streaking of basic proteins on two-dimensional gels
Zinc adaptation and resistance to cadmium toxicity in mammalian cells. Molecular insight by proteomic analysis
To identify proteins involved in cellular adaptive responses to zinc, a
comparative proteome analysis between a previously developed high zinc- and
cadmium- resistant human epithelial cell line (HZR) and the parental HeLa cells
has been carried out. Differentially produced proteins included co-chaperones,
proteins associated with oxido-reductase activities, and ubiquitin. Biochemical
pathways to which these proteins belong were probed for their involvement in
the resistance of both cell lines against cadmium toxicity. Among endoplasmic
reticulum stressors, thapsigargin sensitized HZR cells, but not HeLa cells, to
cadmium toxicity more acutely than tunicamycin, implying that these cells
heavily relied on proper intracellular calcium distribution. The similar
sensitivity of both HeLa and HZR cells to inhibitors of the proteasome, such as
MG-132 or lactacystin, excluded improved proteasome activity as a mechanism
associated with zinc adaptation of HZR cells. The enzyme
4-hydroxyphenylpyruvate dioxygenase was overproduced in HZR cells as compared
to HeLa cells. It transforms 4-hydroxyphenylpyruvate to homogentisate in the
second step of tyrosine catabolism. Inhibition of 4-hydroxyphenylpyruvate
dioxygenase decreased the resistance of HZR cells against cadmium, but not that
of HeLa cells, suggesting that adaptation to zinc overload and increased
4-hydroxyphenylpyruvate removal are linked in HZR cellsComment: in press in Proteomic
Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses
The molecular responses of macrophages to copper-based nanoparticles have
been investigated via a combination of proteomic and biochemical approaches,
using the RAW264.7 cell line as a model. Both metallic copper and copper oxide
nanoparticles have been tested, with copper ion and zirconium oxide
nanoparticles used as controls. Proteomic analysis highlighted changes in
proteins implicated in oxidative stress responses (superoxide dismutases and
peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and
mitochondrial proteins (especially oxidative phosphorylation complex subunits).
Validation studies employing functional analyses showed that the increases in
glutathione biosynthesis and in mitochondrial complexes observed in the
proteomic screen were critical to cell survival upon stress with copper-based
nanoparticles; pharmacological inhibition of these two pathways enhanced cell
vulnerability to copper-based nanoparticles, but not to copper ions.
Furthermore, functional analyses using primary macrophages derived from bone
marrow showed a decrease in reduced glutathione levels, a decrease in the
mitochondrial transmembrane potential, and inhibition of phagocytosis and of
lipopolysaccharide-induced nitric oxide production. However, only a fraction of
these effects could be obtained with copper ions. In conclusion, this study
showed that macrophage functions are significantly altered by copper-based
nanoparticles. Also highlighted are the cellular pathways modulated by cells
for survival and the exemplified cross-toxicities that can occur between
copper-based nanoparticles and pharmacological agents
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
Two-dimensional gel electrophoresis in proteomics: past, present and future
Two-dimensional gel electrophoresis has been instrumental in the birth and
developments of proteomics, although it is no longer the exclusive separation
tool used in the field of proteomics. In this review, a historical perspective
is made, starting from the days where two-dimensional gels were used and the
word proteomics did not even exist. The events that have led to the birth of
proteomics are also recalled, ending with a description of the now well-known
limitations of two-dimensional gels in proteomics. However, the
often-underestimated advantages of two-dimensional gels are also underlined,
leading to a description of how and when to use two-dimensional gels for the
best in a proteomics approach. Taking support of these advantages (robustness,
resolution, and ability to separate entire, intact proteins), possible future
applications of this technique in proteomics are also mentioned
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