38 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
Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis
The solubilizing power of various nonionic and zwitterionic detergents as
membrane protein solubilizers for two-dimensional electrophoresis was
investigated. Human red blood cell ghosts and Arabidopsis thaliana leaf
membrane proteins were used as model systems. Efficient detergents could be
found in each class, i.e. with oligooxyethylene, sugar or sulfobetaine polar
heads. Among the commercially available nonionic detergents, dodecyl maltoside
and decaethylene glycol mono hexadecyl ether proved most efficient. They
complement the more classical sulfobetaine detergents to widen the scope of
useful detergents for the solubilization of membrane proteins in proteomics.Comment: website publisher http://www.interscience.wiley.co
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.
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
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
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
Progress in the definition of a reference human mitochondrial proteome
Owing to the complexity of higher eukaryotic cells, a complete proteome is
likely to be very difficult to achieve. However, advantage can be taken of the
cell compartmentalization to build organelle proteomes, which can moreover be
viewed as specialized tools to study specifically the biology and "physiology"
of the target organelle. Within this frame, we report here the construction of
the human mitochondrial proteome, using placenta as the source tissue. Protein
identification was carried out mainly by peptide mass fingerprinting. The
optimization steps in two-dimensional electrophoresis needed for proteome
research are discussed. However, the relative paucity of data concerning
mitochondrial proteins is still the major limiting factor in building the
corresponding proteome, which should be a useful tool for researchers working
on human mitochondria and their deficiencies.Comment: website publisher http://www.interscience.wiley.co
High expression of antioxidant proteins in dendritic cells: possible implications in atherosclerosis
Dendritic cells (DCs) display the unique ability to activate naive T cells
and to initiate primary T cell responses revealed in DC-T cell alloreactions.
DCs frequently operate under stress conditions. Oxidative stress enhances the
production of inflammatory cytokines by DCs. We performed a proteomic analysis
to see which major changes occur, at the protein expression level, during DC
differentiation and maturation. Comparative two-dimensional gel analysis of the
monocyte, immature DC, and mature DC stages was performed. Manganese superoxide
dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC
stage. This important amount of Mn-SOD is a primary antioxidant defense system
against superoxide radicals, but its product, H(2)O(2), is also deleterious for
cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such
peroxide. Prx1 expression level continuously increased during DC
differentiation and maturation, whereas Prx6 continuously decreased, and Prx2
peaked at the immature DC stage. As a consequence, DCs were more resistant than
monocytes to apoptosis induced by high amounts of oxidized low density
lipoproteins containing toxic organic peroxides and hydrogen peroxide.
Furthermore DC-stimulated T cells produced high levels of receptor activator of
nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes
and DCs. This study provides insights into the original ability of DCs to
express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to
detoxify oxidized low density lipoproteins, and to induce high levels of
receptor activator of nuclear factor kappaB ligand by the T cells they activate
and further emphasizes the role that DCs might play in atherosclerosis, a
pathology recognized as a chronic inflammatory disorder.Comment: cpyright: American Society of Biochemistry and Molecular Biolog