Experiment 08Electrophoresis • B.Sc. / M.Sc.
Determination of Protein Molecular Weight by Discontinuous SDS-PAGE (Laemmli System)
🎯 Aim: To cast a discontinuous polyacrylamide slab gel (stacking and resolving gels), fractionate denatured protein samples alongside a prestained molecular weight ladder, calculate relative mobilities ($R_m$), and determine the subunit molecular weight of an unknown protein.
🔬 Theoretical Principle & Isotachophoresis Mechanics
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) in the Laemmli buffer system resolves proteins purely on the basis of polypeptide chain length (molecular mass):
- Denaturation & Charge Uniformity: The anionic detergent SDS binds cooperatively to denatured polypeptide backbones at a constant stoichiometric ratio of approximately 1.4 g SDS per 1.0 g of protein (~one SDS molecule per two amino acids). SDS imparts an overwhelming, uniform negative charge that masks intrinsic charges, creating an identical charge-to-mass ratio for all proteins. Reducing agents ($beta$-mercaptoethanol or DTT) break covalent disulfide bonds.
- Discontinuous Gel & Isotachophoretic Stacking:
- Stacking Gel ($4% text{Acrylamide, pH } 6.8$): Operates between trailing glycine zwitterions (net charge $approx 0$) and fast leading chloride ions ($text{Cl}^-$). The sharp voltage gradient between $text{Cl}^-$ and glycine compresses all protein samples into an ultra-thin starting zone ($< 1,mutext{m}$) before entering the sieving gel.
- Resolving Gel ($12% text{Acrylamide, pH } 8.8$): At higher pH, glycine ionizes to a fully charged anion ($text{glycine}^-$), outrunning the proteins. The concentrated polyacrylamide molecular mesh then separates proteins strictly by size through sieving.
log M_r = -m cdot R_m + C quad text{where} quad R_m = frac{text{Migration distance of protein band}}{text{Migration distance of Bromophenol Blue dye front}}
Figure 8.1: Discontinuous SDS-PAGE Vertical Slab Gel & Molecular Weight Sizing
📋 Polyacrylamide Gel Casting Formulation Recipes
| Stock Solution | 12% Resolving Gel (10 mL) | 4% Stacking Gel (4 mL) | Role in Polymerization |
|---|---|---|---|
| dH2O | 3.3 mL | 2.7 mL | Solvent / Volume balancing |
| 30% Acrylamide / Bis (29:1) | 4.0 mL | 0.67 mL | Pore-forming monomer matrix (Neurotoxic!) |
| 1.5 M Tris-HCl, pH 8.8 | 2.5 mL | — | Resolving buffer (Glycine ionization) |
| 0.5 M Tris-HCl, pH 6.8 | — | 0.5 mL | Stacking buffer (Glycine zwitterion) |
| 10% (w/v) SDS Stock | 0.1 mL (100 μL) | 0.04 mL (40 μL) | Maintains negative charge and denaturation |
| 10% (w/v) APS (Fresh) | 0.1 mL (100 μL) | 0.04 mL (40 μL) | Free radical polymerization initiator |
| TEMED | 0.01 mL (10 μL) | 0.004 mL (4 μL) | Free radical catalyst / accelerator |
💬 Viva-Voce Questions & Model Answers
Q1: Why does SDS-PAGE require both a stacking gel and a resolving gel?
Without the stacking gel, proteins loaded into sample wells would enter the resolving matrix at different times and depths, producing smeared, diffuse bands. The large pores of the 4% stacking gel and the pH 6.8 discontinuity concentrate all protein molecules into a razor-thin band prior to sieving, ensuring sharp resolution.
Q2: Why are APS and TEMED added immediately before pouring the gel?
Ammonium persulfate (APS) decomposes to yield free persulfate radicals ($text{SO}_4^{bullet-}$). TEMED accelerates this radical generation by electron transfer. Once added, vinyl cross-linking of acrylamide and bis-acrylamide proceeds rapidly; delayed pouring causes premature gelling inside the beaker.
Q3: Why is isopropanol or water-saturated butanol layered on top of the freshly poured resolving gel?
Oxygen in ambient air inhibits free radical polymerization of acrylamide. Layering an immiscible liquid excludes atmospheric oxygen and creates a perfectly flat, horizontal interface for the resolving gel boundary.
