Principle of 1D SDS-PAGE and IEF

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) and isoelectric focusing electrophoresis (IEF) are widely used protein separation techniques in biological research. These methods exploit differences in the electrophoretic behavior of proteins under various conditions to achieve separation and analysis.

 

Principles of 1D SDS-PAGE

1D SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is an electrophoretic technique that separates proteins based on their molecular weight. Its principles include the following aspects:

 

1. Protein Denaturation

SDS is an anionic surfactant that binds strongly to proteins, disrupting their native conformation and causing denaturation. Approximately 1.4g of SDS binds to 1g of protein, forming protein-SDS complexes. These complexes carry a significant negative charge, masking the protein's intrinsic charge. Consequently, the migration of the protein during electrophoresis depends solely on its molecular weight rather than its charge.

 

2. Polyacrylamide Gel Sieving Effect

Polyacrylamide gel serves as a molecular sieve. During electrophoresis, protein-SDS complexes migrate towards the anode under an electric field. The gel's pore size determines the migration speed of proteins of different molecular weights: smaller proteins migrate faster, while larger proteins migrate slower.

 

3. Electrophoresis Process

Protein samples are applied to the top of the gel. Under an electric field, proteins are separated based on their molecular weights. After a specific duration, distinct protein bands form on the gel. By comparing these bands with standard protein markers of known molecular weights, the molecular weights of unknown proteins can be estimated.

 

Principles of IEF

Isoelectric focusing electrophoresis (IEF) separates proteins based on differences in their isoelectric points (pI). The principles of IEF include:

 

1. Isoelectric Point (pI)

Each protein has a specific pH at which its net charge is zero, known as the isoelectric point. Proteins carry a negative charge at pH values above their pI and a positive charge at pH values below their pI.

 

2. Establishment of a pH Gradient

IEF gels contain ampholytes, which form a stable pH gradient within the gel under an electric field. The pH gradually increases from the anode to the cathode.

 

3. Protein Separation Process

As protein samples undergo electrophoresis in the IEF gel, protein molecules migrate under the influence of the electric field. When they reach the pH region corresponding to their pI, their net charge becomes zero, and the electrophoretic force ceases, causing the proteins to focus at their isoelectric points. Hence, IEF can precisely separate proteins with minor pI differences.

 

1D SDS-PAGE and IEF are crucial techniques in protein analysis. 1D SDS-PAGE denatures proteins with SDS and separates them based on molecular weight, while IEF separates proteins by exploiting differences in their isoelectric points. The combination of these techniques provides researchers with detailed information on protein properties and structures, facilitating further biological research and applications.