Two Dimensional Gel Electrophoresis Service

Two-dimensional gel electrophoresis (2-DE) is a crucial technique in proteomics, enabling the separation and analysis of complex protein mixtures from biological samples. This method separates proteins in two stages: isoelectric focusing (IEF), which separates proteins based on their isoelectric point (pI), and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins by their molecular weight (Mr). Consequently, 2-DE can separate thousands of proteins and provide detailed information about their pI and molecular weight. This widely used protein analysis method offers direct visual insights into changes in protein abundance and post-translational modifications (PTMs). It is also applicable in various analyses, including whole proteome analysis, biomarker detection, and drug discovery.

 

Sample Preparation

Effective sample preparation is vital for successful 2-DE analysis. Natural samples must be converted into a physicochemical state suitable for IEF while maintaining the proteins' natural charge and molecular weight. The preparation process varies depending on the type and source of proteins but ideally results in the complete dissolution, decomposition, denaturation, and reduction of proteins in the sample.

 

First Dimension: Isoelectric Focusing (IEF)

In the first dimension, proteins are separated based on their isoelectric point (pI). Proteins, being amphoteric molecules, carry a net charge that depends on the surrounding pH. The pI is the pH at which a protein's net charge is zero. Proteins with a positive net charge migrate towards the cathode, decreasing in positive charge until they reach their pI, while proteins with a negative net charge migrate towards the anode, decreasing in negative charge until they reach their pI.

 

Proteins are loaded onto the basic end of the pH gradient gel, and an electric field is applied. This causes the proteins to separate according to their charge, concentrating at the point where the surrounding pH equals their pI. Larger proteins move more slowly through the gel but eventually catch up with smaller proteins of similar charge.

 

Second Dimension: SDS-PAGE

The second dimension of separation follows IEF and can be performed on a flat or vertical system using plate gels. SDS-PAGE is the electrophoretic method typically used, separating polypeptides based on their molecular weight (Mr). This method involves four steps: gel preparation, equilibration of immobilized pH gradient (IPG) strips in SDS buffer, placement of equilibrated IPG strips on SDS gels, and final electrophoresis.

 

SDS denatures proteins and binds to their main chain at a constant molar ratio. When SDS and reducing agents (such as DTT, which cleaves disulfide bonds) are applied, the proteins unfold into linear chains, and their negative charge is proportional to the length of the polypeptide chain. Polyacrylamide forms a mesh-like matrix suitable for protein separation. During SDS-PAGE, smaller proteins migrate faster due to less resistance.

 

Result Visualization: Staining

Various protein visualization methods are available, with Coomassie Blue staining and silver staining being the most common. Silver staining is a sensitive and non-radioactive method that binds amino acid side chains, primarily thiol and carboxyl groups, to silver ions, which are then reduced to free metallic silver, visualizing protein bands as spots. This method is suitable for detecting low protein levels due to its high sensitivity (in the low ng range).

 

Coomassie Blue staining is simpler and more quantitative than silver staining, suitable for detecting protein bands containing approximately 0.2 μg or more protein. Coomassie dye forms protein-dye complexes through van der Waals attraction. Two types of Coomassie Brilliant Blue dyes, R250 and G-250, are commonly used.

 

Further Protein Analysis

In large studies with thousands of protein spots, detecting new spots or the disappearance of individual spots is challenging. Manual comparison of two gels is also impractical. Therefore, differences are detected, and information is extracted from gels using image acquisition hardware and analysis software. Various 2D gel analysis software, such as Melanie, PDQuest, Progenesis, and REDFIN, are available for applications like mass spectrometry identification.

 

Gel Electrophoresis

Gel electrophoresis technology leverages differences in the size and charge of molecules in a sample. DNA or protein samples are loaded onto a porous gel in an ionic buffer medium. Upon applying a charge, molecules of different sizes and charges travel through the gel at different speeds. The porous gel acts as a molecular sieve, separating larger molecules from smaller ones, with smaller molecules moving faster and larger ones lagging behind. The mobility of particles is also influenced by their charges, and two electrodes with opposite charges pull the molecules towards them based on their charge.

 

Applications of Gel Electrophoresis

Gel electrophoresis is widely used in molecular biology and biochemistry laboratories for various applications, including:

 

1. Separating DNA fragments for DNA fingerprinting in forensic investigations.

2. Analyzing polymerase chain reaction (PCR) results.

3. Analyzing genes associated with specific diseases.

4. Conducting taxonomic studies to distinguish species' DNA.

5. Performing paternity testing using DNA fingerprints.

6. Studying protein structure and function.

7. Analyzing antibiotic resistance.

8. Applying blotting techniques for large molecule analysis.

9. Studying evolutionary relationships by analyzing genetic similarities between populations or species.