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
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)
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
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 of PAGE Three physical effects: Sample concentration effect Molecular sieve effect Charge effect
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
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 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 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 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.)