Serum Protein Electrophoresis

What is Electrophoresis?

Electrophoresis is a technique used to separate macromolecules such as DNA or proteins. It uses positive and negative charges to separate charged particles — opposites attract.

Electrophoresis (Positive)

Molecules migrate toward the negative electrode (cathode)

Electrophoresis (Negative)

Molecules migrate toward the positive electrode (anode).

Agarose or polyacrylamide gels

Acts as a molecular sieve, slowing particle movement.

Inside the Electrophoresis Chamber - Gel

The gel is held in a casting tray and contains pores that allow particles to move slowly toward the opposite charge. It is poured as a hot liquid and solidifies as it cools. Agarose gel is commonly used.

Inside the Electrophoresis Chamber - Comb

The comb is placed into slots in the casting tray before pouring the hot gel. Once the gel solidifies, the comb is removed, leaving small wells for loading samples.

Inside the Electrophoresis Chamber - Wells

The wells are formed when the melted gel solidifies around the teeth of the comb. Wells provide a place to load protein or DNA samples before electrophoresis begins.

Inside the Electrophoresis Chamber - Buffer

The buffer is a solution that conducts electricity. It is poured into the electrophoresis chamber until it just covers the gel. The buffer allows current to flow from the cathode, through the buffer, to the anode, maintaining pH and conductivity.

Protein Electrophoresis Principle

Proteins are forced to migrate through a gel by applying an electric field that pulls on charged molecules.

Positively charged proteins move toward the cathode

Negatively charged proteins move toward the anode.

Functions of Proteins

Enzyme catalysis, Metabolic regulation, Binding and transport (Transferrin, Ceruloplasmin, Hemoglobin), Gene regulation (transcription factors), Immunological defense (antibodies), and Cell structure (structural proteins in membranes and walls).

Properties of Proteins - Charge (Amino Acid Composition)

Proteins are composed of amino acids, each containing a central carbon, an amino group, a carboxylic acid group, a hydrogen, and an R group (side chain).

There are 20 amino acids, each with a different R group

Glutamic acid and aspartic acid

Contribute to a negative charge at physiological pH

Lysine and arginine

Contribute to a positive charge at physiological pH.

Properties of Proteins - Isoelectric Point (pI)

The isoelectric point (pI) is the pH at which a protein has no net charge and will not migrate in an electric field. The direction and extent of migration can be altered using acidic, neutral, or alkaline buffer systems. Proteins with greater charge move faster; those with less charge move slower.

Properties of Proteins - Shape

Proteins have complex three-dimensional folding patterns, and shape affects their mobility. Tightly folded (compact) proteins move faster. Loosely folded (elongated) proteins move slower. Native proteins retain biological activity, while denatured proteins (from detergents, pH extremes, or solvents) lose their shape and function.

Properties of Proteins - Size

The molecular weight of a protein depends on its amino acid composition and number of polypeptide chains. Proteins may have one or several polypeptides. Polypeptides can be identical, similar, or different. The number and type of polypeptides affect mass, size, and shape.

Electrophoresis of Proteins - Factors Affecting Migration

Migration depends on charge, size, and shape of the protein. These factors determine electrophoretic mobility through the gel.

Electrophoresis of Human Serum Proteins

Human serum contains many proteins. Albumin: most abundant serum protein, fastest migration rate (MW ≈ 66.5 kDa).

Gamma globulins (antibodies)

Slowest migration rate (MW range 146-970 kDa). Electrophoretic banding patterns can aid in disease diagnosis, such as abnormal globulin production.

SDS-PAGE Protein Electrophoresis

SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) separates proteins by molecular weight. SDS, a detergent, unfolds proteins and gives them a uniform negative charge. β-Mercaptoethanol (BME) breaks disulfide bonds between subunits. The resulting charge-to-mass ratio allows separation solely by size.

Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide is chemically inert, made in varying concentrations for different pore sizes. Polymerizes chemically (not thermally like agarose). Gels run vertically instead of horizontally. Stacking gel (pH 6.8): large pores, starting zone for protein stacking. Resolving gel (pH 8.8): smaller pores, separates proteins by size.

Buffer System in SDS-PAGE

The buffer system is discontinuous, meaning the buffer in the tank differs in pH from the buffer in the gel. This gradient focuses all proteins into a single narrow band in the stacking gel, which then resolves into distinct bands in the resolving gel. Provides high resolution and clear band definition.

Factors Affecting Protein Band Separation

Migration speed is characteristic of each protein. Proteins are sensitive to pH. Increasing voltage causes faster migration but may melt the gel and reduce band resolution. Increasing buffer ionic strength decreases migration speed.

Bio-Rad Mini-PROTEAN® Tetra Cell System

A next-generation vertical gel electrophoresis system for SDS-PAGE. Runs 1-4 mini gels (7 × 8.5 cm) in about 30 minutes. Accepts both precast and hand-cast gels. Includes sample loading guides and leak-free gel casting. Used for routine protein separation and analysis in laboratories.