Capitulo III Electroforesis Video 15

Concepts of Migration in Electrophoretic Techniques

In this section, the theoretical concepts governing migration processes in electrophoretic techniques are discussed. The movement of particles through a solvent under the influence of an electric field is explored.

Understanding Electroforesis

• Electroforesis involves the movement of components in a mixture based on factors like particle structure, electric charges, sizes, shapes, and the applied electric field.

• Components with negative charges migrate towards the anode while those with positive charges move towards the cathode, resulting in distinct bands or lines representing sample components.

• The force acting on particles in electroforesis includes an electrical force influenced by particle charge and electric field strength. This force is affected by factors such as temperature and ionic strength of the medium.

Factors Affecting Particle Movement

• Various forces contribute to particle movement in an electric field, including static attraction towards charged poles, resistance from the medium opposing attraction forces, and diffusion force causing random motion.

• Particle mobility is represented by electrokinetic mobility determined by factors like effective particle radius and medium viscosity. It defines how particles move concerning intensity and charge.

Types of Electrophoretic Methods

Different types of electrophoretic methods based on support materials are discussed here.

Classification Based on Support Material

• Electrophoresis methods are classified into free electrophoresis conducted in liquid media and zone electrophoresis performed using various solid supports like paper, cellulose acetate, starch, agarose, or polyacrylamide.

Gel Electrophoresis Techniques

This section discusses the use of different gel materials in gel electrophoresis and their impact on molecule separation.

Gel Support Materials

• Gel support using gels improves resolution due to negligible absorption and reduced diffusion.

Agarose Gels

• Agarose gels have variable pore sizes suitable for large molecules like nucleic acids, proteins, and viruses.

Polyester and Polyacrylamide Gels

• Polyester gels are effective for protein and small DNA electrophoresis, while agarose gels are better for larger nucleic acids.

Pore Size Effects

• Pore size impacts migration; restrictive pores hinder smaller molecules but can be beneficial for macromolecules like nucleic acids.

Polyacrylamide Gel Characteristics

• Polyacrylamide gels offer stability across pH, temperature, and ionic strength ranges, mechanical stability, transparency for densitometry quantification, resistance to denaturing agents, and inertness to molecules.

Polymerization Process & Gel Assembly

This section covers the polymerization process of polyacrylamide gels and the assembly steps for gel electrophoresis.

Polyacrylamide Gel Preparation

• Polyacrylamide gels are polymerized using compounds like pyramide and bis-acrylamide with added initiators and stabilizers.

Calculating Pore Size in Gels

• Pore size control in polyacrylamide gels is determined by acrylamide percentage calculations (T%) controlling pore size (T%) and bis-acrylamide concentration (C%).

Impact of Pore Size on Migration

• Pore size affects band migration resolution in various gels based on T% values relative to protein sizes.

Gel Electrophoresis Setup & Considerations

This section details gel electrophoresis setup procedures, considerations for preventing undesirable effects during electrophoresis.

Temperature Effects & Cooling Systems

• Heat generation during electrophoresis can lead to band widening due to increased diffusion; efficient cooling systems prevent these issues.

Gel Assembly Steps

• Assembling a gel involves placing spacers between glass plates, securing with clamps, adding a base with rubber seals to prevent leaks, pouring the polymerization solution without bubbles, inserting combs before solidification.

Electrophoresis Techniques Overview

The section provides an overview of different electrophoresis techniques and equipment used in the process.

Electrophoresis Techniques

• Differentiates between continuous and discontinuous systems in electrophoresis based on gel concentration and pH uniformity.

• Discusses gradient electrophoresis, highlighting its ability to separate proteins of varying sizes effectively.

• Explains native and denaturing electrophoresis methods, emphasizing the preservation or alteration of protein structures.

• Introduces SDS-PAGE as a widely used method for protein separation based on molecular weight using polyacrylamide gels.

SDS-PAGE Technique Details

This section delves into the specifics of SDS-PAGE technique, focusing on gel preparation and protein dissociation.

SDS-PAGE Methodology

• Describes how SDS denatures proteins by disrupting their quaternary, tertiary, and secondary structures for uniform negative charge.

• Explains that in SDS-PAGE, protein mobility is solely determined by molecular weight due to linearized monomers.

Isoelectric Focusing Principles

The discussion centers around isoelectric focusing principles and its application in separating proteins based on their isoelectric points.

Isoelectric Focusing

• Explores how proteins migrate based on their net charges concerning the pH gradient in isoelectric focusing.

Lecture on Gel Electrophoresis Techniques

In this section, the speaker discusses various aspects of gel electrophoresis techniques, including gradient destabilization, immobilized gradients, equipment setup, and detection methods.

Gradient Destabilization and Immobilized Gradients

• The pH gradient destabilizes over time, causing protein displacement.

• Immobilized gradients improve upon traditional gradients by using chemicals like acrylamide to stabilize the pH gradient. This method offers higher stability and resolution with no drift.

Equipment Setup and Gel Types

• Horizontal, vertical, or tube setups are used for electrophoresis. Gels vary in composition based on molecule size and denaturation requirements.

• Basic equipment includes a power source for high voltages, an electrophoresis chamber, and a temperature-controlled bath. Gel strips are placed horizontally for analysis.

Detection Methods

• Proteins or nucleic acids separated via electrophoresis require transfer to immobilized membranes for visualization. Staining methods like Coomassie Blue aid protein detection.

• Radioactive materials or antibodies can be used for specific band detection post-transfer. Enzymes or fluorescent markers enhance visualization efficiency.

Advanced Electrophoresis Techniques

This section delves into advanced electrophoresis techniques such as two-dimensional electrophoresis for enhanced resolution through sequential separations based on different physical properties.

Two-Dimensional Electrophoresis

• Two-dimensional electrophoresis involves two separate runs under varying conditions on the same sample to achieve superior resolution.

• The first dimension separates components based on differential physical properties like charge, shape, size, or isoelectric point.

Electrophoresis Techniques Overview

The discussion covers the separation of components based on different parameters in electrophoretic techniques, leading to maximum resolution. Various combinations of separation modes are highlighted for effective analysis.

Electrophoretic Separation Methods

• Electrophoresis involves separating components based on parameters other than the first one, resulting in maximum resolution.

• Examples include using discontinuous and continuous dimensions for proteins, utilizing gels with different properties in each dimension.

• The most widely used option involves transferring bands to nitrocellulose paper (blotting) for specific identification procedures.

• Strategies like extraction, digestion from gels, followed by sequencing or mass spectrometry analysis are employed for further investigation.

Applications of Two-Dimensional Electrophoresis

This segment explores the applications and complexities associated with two-dimensional electrophoresis techniques in analyzing complex protein mixtures.

Protein Analysis Using Two-Dimensional Electrophoresis

• Electro programs involving bidimensional electrophoresis showcase the complexity of spot patterns and their applications in various fields.

• Applications include separating complex protein mixtures, determining protein composition in different biological samples (e.g., tissues), known as proteomics.

Comparative Proteomic Analysis

The discussion delves into comparative proteomic analysis using two-dimensional electrophoresis as a fundamental tool for studying diverse experimental conditions.

Comparative Proteomic Analysis

• Comparative proteomic analysis aids in comparing protein compositions under varied experimental conditions such as healthy vs. diseased tissues or responses to drugs/toxins.