Electrophoresis and Protein Analysis Fundamentals

General Principles of Electrophoresis

• analysis of the fraction eluted from chromatography columns is carried out by gel electrophoresis

• Electrophoresis is defined as the migration of ions within an electric field.

• It is a widely utilized analytical and preparative technique for the separation of biological macromolecules, particularly proteins and nucleic acids.

• The separation process typically utilizes a fraction eluted from chromatography columns for further analysis via gel electrophoresis.

Quantitative Mechanics of Electrophoretic Migration

• The movement of an ion in an electric field is governed by the laws of electrostatics and fluid dynamics.

• Electric Force ($F_{electric}$): The force exerted on an ion with charge $q$ in an electric field of strength $E$ is expressed as:   $F_{electric} = q \times E$

• Frictional Force ($F_{friction}$): The electrophoretic migration of the ion through a solution is opposed by a frictional force, which is proportional to the velocity ($v$) and the frictional coefficient ($f$):   $F_{friction} = v \times f$

• The frictional coefficient ($f$) depends on several factors:

  • Size of the ion.

  • Shape of the ion.

  • Degree of solvation of the ion.

  • Viscosity of the solution through which the ion is moving.

• Constant Velocity Condition: In a constant electric field, the electric force and frictional force balance each other ($q \times E = v \times f$), meaning the ion moves at a constant characteristic velocity.

• Electrophoretic Mobility ($\mu$): This is defined as the ratio of the velocity to the electric field strength, or the ratio of the charge to the frictional coefficient:   $\mu = \frac{v}{E} = \frac{q}{f}$

Gel Matrix and Separation Media

• Gel electrophoresis is considered one of the most powerful methods for macromolecular separation.

• Gels act as molecular sieves with pores of specific dimensions.

• separates macromolecules based on their size and charge

• Polyacrylamide Gel Electrophoresis (PAGE):

  • Gels are created through the free radical polymerization of acrylamide and the cross-linker N,N’-methylene bisacrylamide.

  • The percentage of acrylamide determines the mesh size (pore size). For example, a 15% SDS-PAGE gel has a smaller mesh size than a 7% SDS-PAGE gel.

  • used to seperate biological macromolecules usually proteins and nucleic acids

• Agarose Gel:

  • Generally used for larger macromolecules, such as nucleic acids.

  • Common concentrations include 1% and 2% agarose. A 2% agarose gel has a smaller mesh size than a 1% agarose gel.

Agarose Gel Electrophoresis for Nucleic Acids

• This specific application is used to separate DNA fragments such as those resulting from restriction digests.

• Fragments are visualised using Ethidium bromide, which intercalates into the DNA.

• Results are captured using a Gel doc system equipped with a CCD camera.

• Typical fragment sizes mentioned include 10 kbps and 1.2 kbps.

• using an electrical field, molecules can be made to move through the gel

• the -ve end (cathode) pushes molecules through the gel, the +ve (anode) pulls molecules through the gel

• small molecules move faster, bigger molecules move slower

• at the end, different sized molecules form distinct bands on the gel

SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)

• This is the most popular application for protein electrophoresis.

• Sodium Dodecyl Sulfate (SDS):

  • A powerful negatively charged detergent.

  • Binds to proteins and unfolds (denatures) them, effectively masking the protein's intrinsic charge with a uniform negative charge.

  • This ensures that proteins migrate toward the positive electrode (anode) solely based on their mass.

• Beta-mercaptoethanol:

  • A reducing agent added to the sample buffer.

  • Functions to break disulfide (S-S) linkages within or between protein subunits.

• Analytical Capabilities:

  • Proteins are separated according to their molecular masses.

  • Relative mobility ($R_{f}$) in these gels varies linearly with the logarithm of the molecular mass.

  • SDS-PAGE can determine molecular mass with an accuracy of 5-10%.

• Components of the Run:

  • Wells: Where protein samples are loaded at the cathode (-) end.

  • Molecular Weight Marker ("Ladder"): A lane containing proteins of known sizes used for calibration.

  • Front of running gel: Indicated by Bromophenol blue dye.

  • relative mobility of proteins in gases vary linearly with the logarithm of their molecular masses

Isoelectric Focusing (IEF)

• Proteins can also be separated based on their native content of acidic and basic residues.

• Isoelectric Point (pI): The specific pH at which a protein's net charge is zero, at this pH the elctrophoretic mobility is zero ($q = 0$ ) no net force from the electric field.

• Mechanism:

  • A pH gradient is established in the gel using a mixture of polyampholytes (small multicharged polymers with varying pI values).

  • At low pH, proteins are positively charged and move toward the cathode.

  • At high pH, proteins are negatively charged and move toward the anode.

  • Proteins stop migrating when they reach the region of the gel where the pH equals their pI.

Two-Dimensional (2D) Gel Electrophoresis

• This technique combines IEF and SDS-PAGE to achieve high-resolution separation.

• Step A: Sample is subjected to isoelectric focusing in a glass tube or strip, separating proteins by their intrinsic charge (pI).

• Step B: The single-lane IEF gel is placed on top of an SDS-polyacrylamide slab gel. SDS is added, and the proteins are run at a right angle to the first dimension, separating them by size (molecular weight).

• This method can resolve thousands of proteins; the only proteins that remain unresolved are those with identical pI and size, which is rare.

Protein Visualization Techniques

1. Coomassie Brilliant Blue (CBB):

   • Most commonly used dye.

   • Binds to basic and hydrophilic amino acids.

   • Sensitivity limit: approximately 25 ng.

2. Silver Staining:

   • Significantly more sensitive than CBB.

   • Binds to specific residues: Asp, Glu, His, Cys, Lys.

   • Sensitivity limit: approximately 0.5 ng.

3. Ponceau Red:

   • Less sensitive (limit: 200 ng).

   • Primarily used to validate the successful transfer of proteins onto nitrocellulose membranes after blotting.

4. Immunoblotting (Western Blotting):

   • Highly specific; requires an antibody that recognizes the protein of interest.

   • Typically uses chemiluminescence for detection.

Immunoblotting (Western Blotting) Protocol

• Rationale: Antibodies cannot effectively penetrate the polyacrylamide gel; therefore, proteins must be transferred to a compatible surface: Nitrocellulose or PVDF (Polyvinylidene difluoride) membranes.

• method:

  • separate proteins by SDS-page

  • detect protein of interest with specific antibodies

  • antibodies cant recognise proteins that are in polyacrylamide gel

  • must transfer proteins onto surface that is compatible with antibodies - nitrocellulose or PVDF membranes

• Assembly of the Transfer Sandwich:

  • The sandwich consists of Whatman filter paper, the polyacrylamide gel, and the Nitrocellulose membrane.

  • A transfer unit (blot chamber) and power supply are used to drive the proteins from the gel onto the membrane.

• Blocking Step:

  • Potential non-specific binding sites on the nitrocellulose are covered with a blocking protein, typically Bovine Serum Albumin (BSA) or milk powder.

• Detection Sequence:

  • Primary Antibody ($1^{st}$ Ab): Recognizes the target protein (e.g., a mouse antibody identifying the "green triangle protein").

  • Secondary Antibody ($2^{st}$ Ab): Recognizes the species-specific primary antibody (e.g., a goat anti-mouse antibody). This secondary antibody is conjugated to an enzyme such as HRP (Horse Radish Peroxidase).

  • Visualisation: Addition of a chemiluminescence substrate causes the HRP to produce light, which is detected by a Gel/Blot doc system.

• Result Analysis: Positions of bands are compared against a molecular weight marker (e.g., bands ranging from 20 kDa to 200 kDa).

Protein Purification and Quantification

• Purification strategies are chosen based on the unique properties of the protein of interest compared to contaminants.

• normally more than one chromatographic column required to obtain pure protein