Describe the process of two-dimensional gel electrophoresis. What are the two dimensions referring to? How is this technique different from conventional gel electrophoresis, and when would it be used?
What will be an ideal response?
In two-dimensional gel electrophoresis, proteins are separated in the first dimension by a version of gel electrophoresis known as isoelectric focusing. In this procedure, proteins are separated exclusively by their charge. A protein's pH environment affects its charge, and every protein has a pH–called the isoelectric point–at which it has neutral charge and cannot move in an electrical field. In isoelectric focusing, proteins migrate through a pH gradient to their isoelectric point, where they stop.
Once isoelectric focusing is complete, protein separation takes place in the second dimension that uses sodium dodecyl sulfate (SDS) gel electrophoresis. SDS is a strong anionic detergent that denatures proteins by disrupting the interactions that keep them folded. Denatured proteins migrate through the gel at a rate determined by their mass; that is, the rate is determined by the number of amino acids they contain. In the SDS gel dimension of two-dimensional gel electrophoresis, each protein has a unique starting point corresponding to its isoelectric point. Proteins with large mass (more amino acids) migrate a short distance in the second dimension, whereas proteins with small mass (fewer amino acids) migrate a greater distance.
One-dimensional gel electrophoresis uses a buffered solution to maintain constant pH throughout the gel, while isoelectric focusing gels contain a pH gradient. Therefore, two-dimensional gel electrophoresis separates proteins based on charge and mass.
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