扬州大学：《生物化学 Biochemistry》课程教学课件（讲稿）Experiments for Biochemsitry 1_Separation of Proteins by Polyacrylamide Gel Electrophoresis（PAGE）

Experiment 7: Separation of Proteins byPolyacrylamideGelElectrophoresis(PAGE)口 PAGE: an electrophoretic method that utilizespolyacrylamide gel as the support medium.口It serves as a vital tool for studying macromoleculessuch as proteins and nucleic acids口The gel is a 3D network structure polymerized fromacrylamide (monomers) and methylene bisacrylamide(cross-linker) in the presence of a catalyst.['meerli:n]-CH2

Experiment 7: Separation of Proteins by Polyacrylamide Gel Electrophoresis (PAGE)  PAGE: an electrophoretic method that utilizes polyacrylamide gel as the support medium.  It serves as a vital tool for studying macromolecules such as proteins and nucleic acids.  The gel is a 3D network structure polymerized from acrylamide (monomers) and methylene bisacrylamide (cross-linker) in the presence of a catalyst. ['meθɪliːn] -CH2

l.PolymerizationofAcrylamidePolyacrylamide gel is formed through the polymerization andcrosslinking of the monomer acrylamide (Acr) and the crosslinkerN,N-methylene-bisacrylamide (Bis) under the action of theaccelerator N,N,N,N-tetramethyl ethylenediamine (TEMED) andthe catalyst ammonium persulfate (AP) or riboflavin (vitamin B2)This 3D network structure gel serves as the support medium forelectrophoresis, which is referred to as polyacrylamide gelelectrophoresis (PAGE)

1. Polymerization of Acrylamide Polyacrylamide gel is formed through the polymerization and crosslinking of the monomer acrylamide (Acr) and the crosslinker N,N-methylene-bisacrylamide (Bis) under the action of the accelerator N,N,N,N-tetramethyl ethylenediamine (TEMED) and the catalyst ammonium persulfate (AP) or riboflavin (vitamin B2). This 3D network structure gel serves as the support medium for electrophoresis, which is referred to as polyacrylamide gel electrophoresis (PAGE)

> PhotopolymerizationCatalysed under light excitation (sunlight or daylight)using riboflavin as the catalyst to produce macroporous gel> Chemical polymerizationAP serves as the initiatorTEMED acts as the acceleratorUsed to produce microporous gel

Photopolymerization Catalysed under light excitation (sunlight or daylight), using riboflavin as the catalyst to produce macroporous gel. Chemical polymerization AP serves as the initiator. TEMED acts as the accelerator. Used to produce microporous gel

Reaction:—CH2—CH—[CH2—CH]—CH2O=oCH,=CH—CCONH2NHNHCH2CH2=CH—CONH2+JCH2NHNHCONH2CH,=CHIC=010—CH2—CH—[CH2—CH]x—CH2-BisAcrpolyacrylamide gel

NH CH2=CH—CONH2 + CH2=CH—C CH2=CH—C —CH2—CH—[CH2—CH]x—CH2— —CH2—CH—[CH2—CH]x—CH2— CONH2 C=O CH2 NH NH O O CONH2 C=O NH CH2 Acr Bis polyacrylamide gel Reaction:

2.Principles ofPAGEThree physical effects:> Sample concentration effect> Molecular sieve effect> Charge effect

2. Principles of PAGE Three physical effects:  Sample concentration effect  Molecular sieve effect  Charge effect

2.PrinciplesofPAGEThree physical effects:> Sample concentration effectarising from the discontinuity of the gel system Discontinuity in gel pore size② Discontinuity in buffer ion composition③ Discontinuity in pH

2. Principles of PAGE Three physical effects:  Sample concentration effect arising from the discontinuity of the gel system. ① Discontinuity in gel pore size ② Discontinuity in buffer ion composition ③ Discontinuity in pH

Discontinuity in gel pore sizeAcr(g)+ Bis(gT(%) =x100%MonomerconcentrationV(ml)Bis(g)x100%C(%) :Degree of crosslinkingAcr(g)+Bis(g)Macroporous gel: T = 3% C= 2.0%Microporous gel: T=7% C=2.5%

① Discontinuity in gel pore size Monomer concentration Degree of crosslinking Macroporous gel: T = 3% C = 2.0% Microporous gel: T = 7% C = 2.5% () () (%) 100% ( ) Acr g Bis g T V ml + = × ( ) (%) 100% () () Bis g C Acr g Bis g = × +

②Discontinuity in buffer ion compositionMacroporous Gel: pH=6.7HCI--->CI-+HLeading ion (Cl-) : exists within the gel layer at any pH value.(fast)Trailing ion (Gly-) : exists in the electrophoresis buffer. (slow)(pI = 6.0, pKαl = 2.34, pKg2 = 9.70)口At pH 6.7, only a minority of glycine is dissociated into Gly ,while the majority remains as Gly口The protein molecule exists in the form of protein- at pH 6.7

②Discontinuity in buffer ion composition • Macroporous Gel: pH=6.7 • Leading ion (Cl⁻) : exists within the gel layer at any pH value.(fast) • Trailing ion (Gly⁻) : exists in the electrophoresis buffer. (slow) (pI = 6.0, pKα1 = 2.34, pKα2 = 9.70)  At pH 6.7, only a minority of glycine is dissociated into Gly⁻, while the majority remains as Gly.  The protein molecule exists in the form of protein⁻ at pH 6.7. HCl Cl H − −+ −−> +

③ Discontinuity in pHpH6.7Macroporous (Concentration) Gel8.9Microporous (Separation) Gel8.3Electrophoresis buffer

③ Discontinuity in pH pH Macroporous (Concentration) Gel 6.7 Microporous (Separation) Gel 8.9 Electrophoresis buffer 8.3

Sample Concentration Effect (Discontinuity)Discontinuity allows for the control of Gly's degree of dissociation,thereby regulating its effective mobilityMobility: Cl-> Protein- > Gly-(Protein is compressed into a thin layer) In the macroporous gel: the effective mobility of Gly is lower than that of allproteins, which is achieved at a pH of 6.7. In the microporous gel: the effective mobility of Gly exceeds that of allproteins, with proteins lagging behind to undergo regular electrophoresisand molecular sieve separation, resulting in multiple zones. (The measuredpH for this condition is actually 9.5.)

Sample Concentration Effect (Discontinuity) Discontinuity allows for the control of Gly's degree of dissociation, thereby regulating its effective mobility. Mobility: Cl⁻ > Protein⁻ > Gly⁻ (Protein is compressed into a thin layer)  In the macroporous gel: the effective mobility of Gly is lower than that of all proteins, which is achieved at a pH of 6.7.  In the microporous gel: the effective mobility of Gly exceeds that of all proteins, with proteins lagging behind to undergo regular electrophoresis and molecular sieve separation, resulting in multiple zones. (The measured pH for this condition is actually 9.5.)