For instance, a 7% polyacrylamide gel has larger pores than a 12% polyacrylamide gel. The size of the pores created in the gel is inversely related to the polyacrylamide percentage (concentration). Protein gel electrophoresis is, therefore, a fundamental step in many kinds of proteomics analysis.Įxample recipe for a traditional polyacrylamide gel: 10% Tris-glycine mini gel for SDS-PAGE: Once separated by electrophoresis, proteins can be detected in a gel with various stains, transferred onto a membrane for detection by western blotting and/or excised and extracted for analysis by mass spectrometry. Proteins with less mass travel more quickly through the gel than those with greater mass because of the sieving effect of the gel matrix.
Thus, when a current is applied, all SDS-bound proteins in a sample will migrate through the gel toward the positively charged electrode. SDS-PAGE separates proteins primarily by mass because the ionic detergent SDS denatures and binds to proteins to make them uniformly negatively charged. Two-dimensional (2D) PAGE separates proteins by native isoelectric point in the first dimension and by mass in the second dimension. Nondenaturing PAGE, also called native-PAGE, separates proteins according to their mass/charge ratio. Denaturing and reducing sodium dodecyl sulfate PAGE (SDS-PAGE) with a discontinuous buffer system is the most widely used electrophoresis technique and separates proteins primarily by mass. Several forms of polyacrylamide gel electrophoresis (PAGE) exist, and each form can provide different types of information about proteins of interest. Polyacrylamide has a smaller pore size and is ideal for separating majority of proteins and smaller nucleic acids. Agarose has a large pore size and is suitable for separating nucleic acids and large protein complexes. These matrices serve as porous media and behave like a molecular sieve. Polyacrylamide and agarose are two support matrices commonly used in electrophoresis. The mobility of a molecule through an electric field will depend on the following factors: field strength, net charge on the molecule, size and shape of the molecule, ionic strength, and properties of the matrix through which the molecule migrates (e.g., viscosity, pore size). Most biological molecules carry a net charge at any pH other than their isoelectric point and will migrate at a rate proportional to their charge density. Both proteins and nucleic acids may be separated by electrophoresis, which is a simple, rapid, and sensitive analytical tool.
Protein electrophoresis is a standard laboratory technique by which charged protein molecules are transported through a solvent by an electrical field.
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