Bioinformatics Solutions Inc.

Gel electrophoresis is an experimental method to separate large molecules like proteins based on size, shape and net charge of the particle. When a molecule is put into an electrical field it will move towards either the positive (cathode) or the negative (anode) electode. If all the species or particles are negative they will all migrate to the anode given an electric field. Proteins are separated by charge and size.

The "gel" in gel electrophoresis is the matrix or medium that the protein is suspended in during the process. The matrix inhibits convective mixing caused by heating that is generated by the electrical current and holds the protein in place after current has been shut off. The matrix can be paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The most commonly used support gels are agarose or polyacrylamide. Agarose, being generally more rigid, is used when separating very large molecules like nucleic acids and large proteins or protein complexes. Polyacrylamide is more flexible and is used for most proteins, peptides and oligonucleotides.

SDS-PAGE (Sodium dodecyl sulphate Polyacrylmide gel electrophoresis) is a experimental method that removes differences in protein shape and electrical charge to allow species to be separated by molecular weight only. SDS is an anionic detergent that disassociates protein into polypeptide chains. The detergent binds to the protein and confers a negative charge that is in proportion to the polypeptide's length. Because of di-sulphide bridging that occurs in protein, an ammount of 2-mercaptoethanol or dithiothreitol is introduced to remove protein chain self-association. What is left are charged random coils of amino acids of various lengths which will separate by PAGE on their molecular weight alone.

With 2D gel electrophoresis there are 2 steps. The first is isoelectric focusing where the proteins arranged by their native charge. The second step is SDS-PAGE that is performed on the charge separated resultant proteins from step one. Isoelectric focusing is carried out using polyacrylamide gel that has a pH gradient. The protein mixture is loaded in the center of one side, where the pH is neutral. A voltage is applied across the gel and the proteins migrate to their isoelectric position. The isoelectric position is the pH where the net charge on the protein is zero. This method allows proteins to be separated by a number of charges that the protein has naturally. This results in the proteins being lined-up roughly along one edge of the gel, parallel to the pH gradient.
For the next step the voltage will be applied from top to bottom instead of side to side. The gel will be soaked in SDS (Sodium dodecyl sulphate) that will denature the proteins in the gel and allow the proteins to migrate to the bottom of the gel based only on the length of its amino acid chain.

	News \| Products Downloads Support User Comments Corporate
De Novo Sequencing Protein ID inChorus PTM Seq. Tag Search Raw Data Download Demo Request Prices Tech Support Our Research User Comments	Superior de novo sequencing and protein ID Software PEAKS Gel Electrophoresis Gel electrophoresis is an experimental method to separate large molecules like proteins based on size, shape and net charge of the particle. When a molecule is put into an electrical field it will move towards either the positive (cathode) or the negative (anode) electode. If all the species or particles are negative they will all migrate to the anode given an electric field. Proteins are separated by charge and size. The Matrix The "gel" in gel electrophoresis is the matrix or medium that the protein is suspended in during the process. The matrix inhibits convective mixing caused by heating that is generated by the electrical current and holds the protein in place after current has been shut off. The matrix can be paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The most commonly used support gels are agarose or polyacrylamide. Agarose, being generally more rigid, is used when separating very large molecules like nucleic acids and large proteins or protein complexes. Polyacrylamide is more flexible and is used for most proteins, peptides and oligonucleotides. SDS-PAGE Safety tip: Don't eat the gel!! SDS-PAGE (Sodium dodecyl sulphate Polyacrylmide gel electrophoresis) is a experimental method that removes differences in protein shape and electrical charge to allow species to be separated by molecular weight only. SDS is an anionic detergent that disassociates protein into polypeptide chains. The detergent binds to the protein and confers a negative charge that is in proportion to the polypeptide's length. Because of di-sulphide bridging that occurs in protein, an ammount of 2-mercaptoethanol or dithiothreitol is introduced to remove protein chain self-association. What is left are charged random coils of amino acids of various lengths which will separate by PAGE on their molecular weight alone. 2D Gel Electrophoresis With 2D gel electrophoresis there are 2 steps. The first is isoelectric focusing where the proteins arranged by their native charge. The second step is SDS-PAGE that is performed on the charge separated resultant proteins from step one. Isoelectric focusing is carried out using polyacrylamide gel that has a pH gradient. The protein mixture is loaded in the center of one side, where the pH is neutral. A voltage is applied across the gel and the proteins migrate to their isoelectric position. The isoelectric position is the pH where the net charge on the protein is zero. This method allows proteins to be separated by a number of charges that the protein has naturally. This results in the proteins being lined-up roughly along one edge of the gel, parallel to the pH gradient. For the next step the voltage will be applied from top to bottom instead of side to side. The gel will be soaked in SDS (Sodium dodecyl sulphate) that will denature the proteins in the gel and allow the proteins to migrate to the bottom of the gel based only on the length of its amino acid chain.