Polyacrylamide GelElectrophoresis(PAGE)
Polyacrylamide Gel Electrophoresis (PAGE)
1.PrinciplesElectrophoresis is a method used to separatemacromolecules based on their charge, binding affinity,and sizeunderanelectricfieldPolyacrylamidegel electrophoresis (PAGE) isapplicable to separate biological macromolecules,primarily proteins or nucleic acids, based on theirelectrophoreticmobility.The most commonly used PAGE for quantitative proteinanalysis is Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE)
1.Principles Electrophoresis is a method used to separate macromolecules based on their charge, binding affinity, and size under an electric field. Polyacrylamide gel electrophoresis (PAGE) is applicable to separate biological macromolecules, primarily proteins or nucleic acids, based on their electrophoretic mobility. The most commonly used PAGE for quantitative protein analysis is Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
1.1PolyacrylamideGelThe polyacrylamide gel forms by polymerizingacrylamide and a crosslinking agent, i.e., N, N'-methylene-bis-acrylamideItdoesnotreactwithproteinsandconsistsofporesandchannels that allow the protein to move through itCH业
1.1 Polyacrylamide Gel The polyacrylamide gel forms by polymerizing acrylamide and a crosslinking agent, i.e., N, N’- methylene-bis-acrylamide. It does not react with proteins and consists of pores and channels that allow the protein to move through it
1.2PrinciplesofPAGEPAGE is based on the principle that charged particlesmigrate to the electrode of the opposite sign under theinfluenceofanelectricfield.In PAGE, the sample dissolves into a buffer containingdenaturing agents, glycerol, bromothymol blueAn anionic detergent, sodium dodecyl sulfate, helpsdenature the sample protein and binds to the side chainof amino acids at one SDs anion per two residues.Therefore, the negative charge of SDS results in a netnegative charge in the protein sample
1.2 Principles of PAGE PAGE is based on the principle that charged particles migrate to the electrode of the opposite sign under the influence of an electric field. In PAGE, the sample dissolves into a buffer containing denaturing agents, glycerol, bromothymol blue. An anionic detergent, sodium dodecyl sulfate, helps denature the sample protein and binds to the side chain of amino acids at one SDS anion per two residues. Therefore, the negative charge of SDS results in a net negative charge in the protein sample
1.2PrinciplesofPAGEGlycerolhelpstoincreasethedensity oftheprotein sampleso that the protein sample falls at the bottom of the gel ratherthan floating or mixing with the running buffer.Bromothymol blue helps to visualize the protein sample in agel during electrophoresis.When the electric current passes through the electrophoreticchamber, the protein-SDs complex starts moving toward theanode.The gel with a larger pore size (containing 4-8% acrylamide)permits higher molecular weight molecules to move fasterthrough the gel,while the gel with a smaller pore size(containing 12-20% acrylamide) restricts the migration oflarger molecules allowing only passage of smaller molecules
1.2 Principles of PAGE Glycerol helps to increase the density of the protein sample so that the protein sample falls at the bottom of the gel rather than floating or mixing with the running buffer. Bromothymol blue helps to visualize the protein sample in a gel during electrophoresis. When the electric current passes through the electrophoretic chamber, the protein-SDS complex starts moving toward the anode. The gel with a larger pore size (containing 4-8% acrylamide) permits higher molecular weight molecules to move faster through the gel, while the gel with a smaller pore size (containing 12-20% acrylamide) restricts the migration of larger molecules allowing only passage of smaller molecules