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Transcript  Page 1 TWO-DIMENSIONAL GEL ELECTROPHORESIS Compiled by Dr. V. Magendira Mani., M.Sc., M.Phil., Ph.D., Assistant Professor, PG & Research Department of Biochemistry, Islamiah College (Autonomous), Vaniyambadi, Vellore District  –   635751, Contact : +91 9486000227 Mail:  Download Science at:  Page 2 Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis (2-D electrophoresis) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This technique separate proteins in two steps, according to two independent properties: the first-dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW). In this way, complex mixtures consisted of thousands of different proteins can be resolved and the relative amount of each protein can be determined. The procedure involves placing the sample in gel with a pH gradient, and applying a potential difference across it. In the electrical field, the protein migrates a long the pH gradient, until it carries no overall charge. This location of the protein in the gel constitutes the apparent pI of the protein. There are two alternatives methods to create the pH gradient - carrier ampholites and immobilized pH gradient (IPG) gels. In the Maiman Institute for Proteome Research the IEF is performed with commercial IPGs for highly reproducible results.  Page 3 The IEF is the most critical step of the 2-D electrophoresis process. The proteins must be solubilize without charged detergents, usually in high concentrated urea solution, reducing agents and chaotrophs. To obtain high quality data it is essential to achieve low ionic strength conditions before the IEF itself. Since different types of samples differ in their ion content, it is necessary to adjust the IEF buffer and the electrical profile to each type of sample. The separation in the second dimension by molecular size is performed in slab SDS- PAGE. Twelve parallel gels can be separated in a fixed temperature to minimize the separation variations between individual gels. For IEF and second dimension electrophoresis, the sample should be free of salts and ions, and they must be removed by dialysis Visualization After electrophoresis the gel is stained to visualize the separated proteins. Commonly used stains are Coomassie Brilliant Blue or SYPRO Ruby or silver stain. Different proteins will appear as distinct spot within the gel. Coomassie Brilliant Blue or SYPRO Ruby are compatible with Mass Spectrometry. Coomassie Brilliant Blue has detection limit about 10ng of proteins per spot and the gel images of spots can be captured by scanning densitometer which operate in visible light. SYPRO Ruby can detect 1ng of proteins per spot and since it is fluorescent, the spots are visualized by a fluorescent imager. Silver stain can detect spots containing proteins less than 1 ng and is the most sensitive non  –   radioactive protein visualization method. Laser devices for image capturing are useful for fluorescently stained gels. Analysis The images can be further analyzed using image  –  analysis softwares. These softwares quantify proteins spots, match images and compare corresponding spots intensities of related gels, prepare gel data reports, remove background patterns, and integrate image information to databases. Alternately the proteins separated can be obtained from the gel can be analyzed by MS for protein identification. 2D- PAGE can be used to study differential protein expression by comparing images from 2D  –  PAGE gels samples labeled with stable isotopes or fluorescent dyes.  Page 4 Advantages    Using 2D-PAGE, 100  –1000’s of polypeptides can be analyzed in a single run.    2D-PAGE is the primary technique for proteomics work. It separates the complex mixture of samples using two different properties of the proteins.    Proteins separated on 2D-PAGE are nearly pure, therefore, they can be excised and used in the Edman degradation method of protein sequencing and for mass spectrometric analysis.    2D-PAGE images can also be used to detect certain post-translational modifications like glycosylation, phosphorylation, etc..    2-DE has been used for over three decades and still remains the main method for separation, comparison, and detection of quantitative changes in biomarker expression levels in Neurodegenerative Disease Disadvantages    The disadvantages may include large amount of sample handling, less reproducibility, difficulty to separate low abundance proteins, acidic and basic proteins, very large and very small proteins and hydrophobic proteins. Also not automated for high throughput analysis. 2D-PAGE has limited dynamic range.    2-DE technique is a time-consuming and labor-intensive process.    Conventional 2-DE is restricted to the detection of denatured proteins in the size range of 10~200 kDa at pH 3.5~11.5.   Edited by Dr. V. Magendira Mani Assistant professor of Biochemistry Islamiah College (Autonomous) Vaniyambadi  –   635751 Compiled on 04-02-2018


Mar 12, 2018
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