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More electrophoresis protocols

SDS-PAGE (PolyAcrylamide Gel Electrophoresis)
(Davidson college)
Molecular biology

One-dimensional SDS gel electrophoresis of proteins.
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.1.)
One-dimensional SDS gel electrophoresis of proteins.

Gallagher SR.

Hoefer Pharmacia Biotech, San Francisco, California, USA.

Electrophoresis is used to separate complex mixtures of proteins, to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in the gel matrix; pore size decreases with higher acrylamide concentrations. The combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit contains protocols that gives the standard Laemmli method for discontinuous gel electrophoresis under denaturing conditions, and the standard method for full-size gels is adapted for the minigel format. Minigels provide rapid separation but give lower resolution. Several alternate protocols are provided for specific applications, including electrophoresis of peptides and small proteins, continuous SDS-PAGE, ultrathin gels, multiple single-concentration gels, gradient gels, multiple gradient gels, and multiple gradient minigels.

Protein Ladder
Storage of protein marker ladder

Electrophoresis technique books
(Biowww bookshelf)
Electrophoresis technique related books

protein purification from acrylamide gel
need protocol to purify a protein band from a SDS-PAGE and then elute it

Laemmli buffer composition for diluting protein
composition of Laemmli buffer for protein electrophoresis.

no bands in SDS-PAGE
Protein electrophoresis problem

Capillary electrophoresis of peptides and proteins using isoelectric buffers.
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.13.)
Righetti PG, Bossi A, Gelfi C.

University of Verona, Verona, Italy.

Capillary electrophoresis in acidic isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and no interaction of macromolecules with the untreated wall ensues. In addition, because of the low conductivity of quasi-stationary isoelectric buffers, high voltage gradients can be applied (up to 800 V/cm), permitting fast peptide analysis with a high resolving power and minimal diffusional peak spreading.

Capillary electrophoresis of proteins and peptides
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.9.)
Burgi D, Smith AJ.

Genomyx, Foster City, California, USA.

Capillary electrophoresis (CE) is a high-resolution technique for the separation of a wide variety of molecules of biological interest such as metabolites, drugs, amino acids, nucleic acids, and carbohydrates. This unit focuses on the use of CE to separate proteins and peptides based on charge-to-mass ratios. Separation of proteins based on their isoelectric points is described along with a protocol for optimizing the separation conditions for a given protein. CE is also used for separations of peptides on an analytical scale and as a micropreparative technique--with either multiple separations that are pooled or a single, larger-scale separation--for the isolation of peptides from a protease digestion. In most of these examples the same capillary column can be used for all the separations. Only changes in buffer composition, ionic strength, and the presence or absence of additives are required for each specific application.

One-dimensional electrophoresis using nondenaturing conditions
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.3.)
Gallagher SR.

Hoefer Pharmacia Biotech, San Francisco, California, USA.

Nondenaturing or "native" electrophoresis--i.e., electrophoresis in the absence of denaturants such as detergents and urea--is an often-overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Two protocols are presented in this unit: continuous PAGE, which is highly flexible, permitting cationic and anionic electrophoresis over a full range of pH, and discontinuous PAGE, which is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration.

Transverse urea-gradient gel electrophoresis
(Curr Protoc Protein Sci. 2001 May;Chapter 7:Unit 7.4.)
Goldenberg DP.

University of Utah, Salt Lake City, Utah, USA.

Monitoring the cooperative unfolding transition induced when a protein is exposed to elevated temperature or a chemical denaturant is an important strategy for characterizing the conformational properties of a globular protein. This transition may be analyzed quantitatively by a variety of spectroscopic techniques, but a simpler alternative is described in this unit: urea-gradient gel electrophoresis. The pattern produced in the resulting gel can be used to estimate both the free energy change for unfolding and the rate of the unfolding transition. In addition, the technique can help identify either covalent or conformational heterogeneity in a protein sample. Because urea-gradient gel patterns are sensitive to several parameters, including hydrodynamic volume, net charge, and conformational stability, the technique can be particularly useful for comparing two forms of a protein, e.g., a natural form and the product of recombinant bacteria.

Protein profiling using two-dimensional difference gel electrophoresis (2-D DIGE)
(Curr Protoc Protein Sci. 2003 Feb;Chapter 22:Unit 22.2.)
Lilley KS.

University of Cambridge, Cambridge, United Kingdom.

2D-DIGE relies on pre-electrophoretic labeling of samples with one of three spectrally distinct fluorescent dyes, followed by electrophoresis of all samples in one gel. The dye-labeled samples are then viewed individually by scanning the gel at different wavelengths, which circumvents problems with spot matching between gels. Image analysis programs can then be used to generate volume ratios for each spot, which essentially describe the intensity of a particular spot in each test sample, and thus enable expression differences to be identified and quantified. This unit describes the DIGE procedure in terms of sample preparation from various types of cells, labeling of proteins, and points to consider in the downstream processing of fluorescently labeled samples.

Two-dimensional gel electrophoresis
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.4.)
Harper S, Mozdzanowski J, Speicher D.

The Wistar Institute, Philadelphia, Pennsylvania, USA.

Two-dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures. Variations of the most common two-dimensional technique are described in this unit, namely isoelectrofocusing (IEF) and SDS-PAGE. This unit also includes support protocols describing pI standards and pH profile measurements, casting Immobiline gels, preparation of tissue culture cells and solid tissues for isoelectricfocusing, preparation of molecular weight standards for two-dimensional gels, and two-dimensional protein databases.

Preparing protein extracts for quantitative two-dimensional gel comparison.
(Curr Protoc Protein Sci. 2004 Aug;Chapter 22:Unit 22.4.)
Chevallet M, Tastet C, Luche S, Rabilloud T.

Départment Résponse et Dynamique, Cellulaire/BioEnergetique Cellulaire et Pathologique, Commissariat à l'Energie Atomique, Grenoble, France.

This unit describes basic protocols for efficient and reproducible protein solubilization from a variety of biological samples, including cultured animal cells and tissues, plant cells and tissues, bacteria, nuclei, other subcellular organelles, plasma, serum, and other biological fluids. The optimized extraction process is strongly sample-dependent and cannot be described for every type of sample. Instead, typical protocols are provided as general guidelines and illustrate good starting points for sample-preparation optimization. These solubilization procedures take into account the constraints brought by two-dimensional electrophoresis and are thus well suited for proteomic approaches.

Protein charge determination
(Curr Protoc Protein Sci. 2005 Sep;Chapter 2:Unit 2.10)
Winzor DJ.

University of Queensland, Brisbane, Queensland.

The most popular current method of determining protein valence entails the calculation of net charge from amino acid sequence/composition. However, the inaccuracy of that approach was recognized long before the advent of the protein data banks and computer programs to facilitate its adoption. Capillary zone electrophoresis affords the simplest and most economical procedure for obtaining a reliable estimate of the net charge of a protein in the buffer system of interest. This unit explains the major pitfalls in the calculation of net charge from protein sequence data.

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