What Are the Types, Principles, and Methods of Protein Electrophoresis?
Protein electrophoresis is a technique that separates proteins under an electric field based on their size, charge, and shape. Below are common types of protein electrophoresis, their principles, and experimental methods:
Continuous SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)
1. Principle
SDS, an anionic surfactant, binds to proteins, linearizing them and giving a uniform negative charge. Migration depends on size, not charge.
2. Method
(1) Sample Preparation: Mix protein with SDS buffer and boil for 5 min.
(2) Loading: Add samples to gel wells.
(3) Electrophoresis: Apply constant voltage for migration.
(4) Staining & Destaining: Use Coomassie Blue or silver stain, then destain to visualize bands.
Discontinuous SDS-PAGE
1. Principle
Uses stacking and separating gels of different concentrations, improving resolution.
2. Method
Similar to continuous SDS-PAGE but requires careful gel layering.
Native-PAGE
1. Principle
No SDS is used; migration depends on size, charge, and shape.
2. Method
Follows SDS-PAGE steps but excludes SDS; staining remains the same.
Blue Native-PAGE
1. Principle
Similar to Native-PAGE, but Coomassie Blue is added to impart a negative charge to proteins.
2. Method
Same as Native-PAGE but includes Coomassie Blue.
Two-Dimensional Electrophoresis (2D-PAGE)
1. Principle
Combines isoelectric focusing (IEF) and SDS-PAGE for high-resolution separation.
2. Method
(1) First Dimension (IEF): Proteins separate based on isoelectric points (pI) in a pH gradient.
(2) Second Dimension (SDS-PAGE): IEF gel strip is processed in SDS and electrophoresed based on molecular weight.
(3) Staining & Analysis: Stain with Coomassie Blue or silver stain, scan, and analyze protein spots.
Isoelectric Focusing (IEF)
1. Principle
Proteins migrate in a pH gradient until reaching their pI, where net charge is zero.
2. Method
(1) Prepare a pH gradient using carrier ampholytes.
(2) Load sample into a polyacrylamide gel.
(3) Apply voltage to focus proteins at their pI.
Capillary Isoelectric Focusing (cIEF)
1. Principle
A variant of IEF where a pH gradient is formed in a capillary or gel via electrophoresis of carrier ampholytes.
2. Method
(1) Load sample with ampholytes into a capillary or gel.
(2) Apply voltage to establish the pH gradient.
(3) Detect focused protein bands via UV or other methods.
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