10,155 research outputs found
Silver Staining of 2D Electrophoresis 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. For its
use in proteomics, two important additional features must be considered,
compatibility with mass spectrometry and quantitative response. Both features
are discussed in this chapter, and optimized silver staining protocols are
proposed.Comment: arXiv admin note: substantial text overlap with arXiv:0904.353
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
Power and limitations of electrophoretic separations in proteomics strategies
Proteomics can be defined as the large-scale analysis of proteins. Due to the
complexity of biological systems, it is required to concatenate various
separation techniques prior to mass spectrometry. These techniques, dealing
with proteins or peptides, can rely on chromatography or electrophoresis. In
this review, the electrophoretic techniques are under scrutiny. Their
principles are recalled, and their applications for peptide and protein
separations are presented and critically discussed. In addition, the features
that are specific to gel electrophoresis and that interplay with mass
spectrometry (i.e., protein detection after electrophoresis, and the process
leading from a gel piece to a solution of peptides) are also discussed
Sweet silver: A formaldehyde-free silver staining using aldoses as developing agents, with enhanced compatibility with mass spectrometry
Protein detection methods after electrophoresis have to be sensitive,
homogeneous, and not to impair downstream analysis of proteins by MS. Speed,
low cost, and user friendliness are also favored features. Silver staining
combines many of these features, but its compatibility with MS is limited. We
describe here, a new variant of silver staining that is completely
formaldehyde-free. Reducing sugars in alkaline borate buffer are used as
developers. While keeping the benefits of silver staining, this method is shown
to afford a much better performance in terms of compatibility with MS, both in
PMF by MALDI and in LC/ESI/MS/MS
Improved mass spectrometry compatibility is afforded by ammoniacal silver staining
Sequence coverage in MS analysis of protein digestion-derived peptides is a
key issue for detailed characterization of proteins or identification at low
quantities. In gel-based proteomics studies, the sequence coverage greatly
depends on the protein detection method. It is shown here that ammoniacal
silver detection methods offer improved sequence coverage over standard silver
nitrate methods, while keeping the high sensitivity of silver staining. With
the development of 2D-PAGE-based proteomics, another burden is placed on the
detection methods used for protein detection on 2-D-gels. Besides the classical
requirements of linearity, sensitivity, and homogeneity from one protein to
another, detection methods must now take into account another aspect, namely
their compatibility with MS. This compatibility is evidenced by two different
and complementary aspects, which are (i) the absence of adducts and artefactual
modifications on the peptides obtained after protease digestion of a protein
detected and digested in - gel, and (ii) the quantitative yield of peptides
recovered after digestion and analyzed by the mass spectrometer. While this
quantitative yield is not very important per se, it is however a crucial
parameter as it strongly influences the S/N of the mass spectrum and thus the
number of peptides that can be detected from a given protein input, especially
at low protein amounts. This influences in turn the sequence coverage and thus
the detail of the analysis provided by the mass spectrometer.Comment: website publisher http://www.interscience.wiley.co
Two-dimensional gel electrophoresis in proteomics: A tutorial
Two-dimensional electrophoresis of proteins has preceded, and accompanied,
the birth of proteomics. Although it is no longer the only experimental scheme
used in modern proteomics, it still has distinct features and advantages. The
purpose of this tutorial paper is to guide the reader through the history of
the field, then through the main steps of the process, from sample preparation
to in-gel detection of proteins, commenting the constraints and caveats of the
technique. Then the limitations and positive features of two-dimensional
electrophoresis are discussed (e.g. its unique ability to separate complete
proteins and its easy interfacing with immunoblotting techniques), so that the
optimal type of applications of this technique in current and future proteomics
can be perceived. This is illustrated by a detailed example taken from the
literature and commented in detail. This Tutorial is part of the International
Proteomics Tutorial Programme (IPTP 2)
Comparision of staining methods for two dimensional electrophoresis gel resolved with Puntius javanicus liver proteome
The aim of this study was to compare the various staining methods based on commassie briliant blue and silver nitrate stain for the two dimensional gel electrophoresis resolved with Puntius javanicus liver proteome. The staining methods were selected base on the previous report about their compatibility with the mass spectrometry analysis. Silver staining method is known as the most sensitive method to visualize the maximum number of protein spots resolved in 2D gel but it is less sensitive(incompatible) toward mass spectrometry detection. Results of this study showed that a modified staining method using colloidal coomassie blue G-250 (CCB) is roughly similarly sensitive but lower protein spot detected compared with silver staining (SS) as indicated at the number of 303±26 and 693±14of protein resolved in both types of stained gels. The conventional methods of staining using commassie brilliant blue G-250 and R-250 only detected less number of protein spots(128±17and 78±11, respectively) compared to modified CCB staining method. As the commasie brilliant blue stain wasis known to be a very sensitive for mass spectrometry detection, the modified method of CCB was selected for further study on Puntius javanicus liver proteome
Fully denaturing two-dimensional electrophoresis of membrane proteins: a critical update
The quality and ease of proteomics analysis depends on the performance of the
analytical tools used, and thus of the performances of the protein separation
tools used to deconvolute complex protein samples. Among protein samples,
membrane proteins are one of the most difficult sample classes, because of
their hydrophobicity and embedment in the lipid bilayers. This review deals
with the recent progresses and advances made in the separation of membrane
proteins by 2-DE separating only denatured proteins. Traditional 2-D methods,
i.e., methods using IEF in the first dimension are compared to methods using
only zone electrophoresis in both dimensions, i.e., electrophoresis in the
presence of cationic or anionic detergents. The overall performances and fields
of application of both types of method is critically examined, as are future
prospects for this field
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
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