扬州大学：《生物化学 Biochemistry》课程教学课件（讲稿）Experiments for Bichemistry 2_Polyacrylamide Gel Electrophoresis（PAGE）

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

1.3EssentialsofelectrophoresisGelplate:theplatethatholdsthepolymerized gel inanelectrophoresischamber.Electrophoresis chamber:Polyacrylamidegel ispackedwithin a running buffer, and electrophoresis is carried out.Power supply:It is essential to create an electricfield

1.3 Essentials of electrophoresis  Gel plate: the plate that holds the polymerized gel in an electrophoresis chamber.  Electrophoresis chamber: Polyacrylamide gel is packed within a running buffer, and electrophoresis is carried out.  Power supply: It is essential to create an electric field

1.4ProcedureinvolvedinPAGE1.4.1SamplepreparationThesamplecanbeeitherproteinornucleicacidThemixingof sampleoccurs inaloadingbuffercontainingdenaturing agents (SDS),I,andbromothymolblueUnlikeglycerol,protein, urea can help as a denaturing agent for nucleic acid.Heating the samples with denaturing agents for 5-10 minutesfurtherenhancesthedenaturation

1.4 Procedure involved in PAGE 1.4.1 Sample preparation  The sample can be either protein or nucleic acid.  The mixing of sample occurs in a loading buffer containing denaturing agents (SDS), glycerol, and bromothymol blue. Unlike protein, urea can help as a denaturing agent for nucleic acid.  Heating the samples with denaturing agents for 5-10 minutes further enhances the denaturation

1.4.2Preparation of polyacrylamidegelIt comprises acrylamide, bisacrylamide, optional denaturant (SDsfor protein and Urea for nucleic acid), and buffer with adjusted pH.All of thereagents arecombined except TEMED.TEMED is mixedwhen the gel is ready to pour.Two types of gels, i.e.,stacking gel pH 6.88andseparatinggelpH8.8are prepared. Furthermore, at first, separating gel is poured, and overit stacking gel is run, ensuring all of the sample proteins arrive at theseparatinggel simultaneously.Afterpolymerization,thecombisinsertedwithinthestackinggellayeronaglassplate.Thepolymerized gel is thenknown as a‘gel cassette

1.4.2 Preparation of polyacrylamide gel  It comprises acrylamide, bisacrylamide, optional denaturant (SDS for protein and Urea for nucleic acid), and buffer with adjusted pH.  All of the reagents are combined except TEMED. TEMED is mixed when the gel is ready to pour.  Two types of gels, i.e., stacking gel pH 6.8 and separating gel pH 8.8, are prepared. Furthermore, at first, separating gel is poured, and over it stacking gel is run, ensuring all of the sample proteins arrive at the separating gel simultaneously.  After polymerization, the comb is inserted within the stacking gel layer on a glass plate.  The polymerized gel is then known as a ‘gel cassette.’

1.4.3ElectrophoresisDepending onthe sampletype,theuseofdifferent buffersystemscan help to run polyacrylamide gel electrophoresis.Thebuffer used at the cathode or anode might bethe same ordifferent.Duringelectrophoresis,thegel cassettewasremovedfromthecastingstandandwasplacedintheelectrodeassembly.Itislateronfixed intheclampstand.Then,1xrunningbufferispouredintotheelectrophoresischamberEachwell isthenloaded with aprotein sample.Similarly,markerprotein is also loaded into a single well of gel.Thetank isthencoveredwitha lid,and the samplecan run at3OmAforabout1hour

1.4.3 Electrophoresis  Depending on the sample type, the use of different buffer systems can help to run polyacrylamide gel electrophoresis.  The buffer used at the cathode or anode might be the same or different.  During electrophoresis, the gel cassette was removed from the casting stand and was placed in the electrode assembly. It is later on fixed in the clamp stand.  Then, 1× running buffer is poured into the electrophoresis chamber.  Each well is then loaded with a protein sample. Similarly, marker protein is also loaded into a single well of gel.  The tank is then covered with a lid, and the sample can run at 30mA for about 1 hour

NegativeElectrodeLoadsampleshereBufferweEwellwalStacking GelRunning Gelbigproteins++Glass++PlatesPositive++ElectrodeSpacersmallproteinsBufferAnillustrationofanapparatususedfor SDSPAGE