LECTURE 12

Biochemical Techniques in Protein Separation

Definition: PAGE is a common method in biochemistry for separating proteins based on size.
Visual Reference: Consult Chapter 5.2 of the Biochemistry core textbook, Figure 5.8 for a graphical representation.

Sodium Dodecyl Sulfate (SDS):
- Chemical formula: CH3-(CH2)11-SO4–Na+
- Characteristics: Dual nature as ionic (hydrophilic head) and hydrophobic (hydrophobic tail).
Function of SDS:
- Forms micelles in aqueous solutions, providing uniform charge distribution among proteins.

At neutral pH, SDS interacts with denatured polypeptide chains, forming complexes.
- Typical Ratio: 1.4 g SDS per g of globular protein; higher for membrane proteins (up to 2 g).
Each protein is converted into a linear denatured polypeptide complexed with SDS.
- Charge: Each complex carries a uniform negative charge.
- Size Relation: Size of the complex is approximately proportional to the protein's molecular weight.

Denaturation: SDS denatures and imparts a uniform charge to proteins regardless of their original charge.
- This guarantees separation based purely on size in SDS-PAGE.
Molecular Weight Estimation: SDS-PAGE allows for the assessment of protein molecular weights based on their mobility in the gel.

SDS does not disrupt disulfide bridges; hence, connected subunits move as a single entity.
- Effect on Mobility: Mobility is affected if S-S bonds are present; reducing agents can cleave these bonds, enhancing separation.
Common Reducing Agents:
- β-mercaptoethanol
- Dithiothreitol (DTT)
- Tris (2-carboxethyl) phosphine (TCEP)

Visual Representation: Proteins are separated into distinct bands in the gel, which are not initially visible without staining.
- Staining process: Gel is dyed, and excess dye removed to highlight protein locations.

The charge of a protein is dependent on pH:
- At pH = pI, net charge of the protein is zero.
- The precise pI curve is unique to each protein's structure and characteristics.

Isoelectric Focusing: Separation is based on the protein's pI, using a gradient from low to high pH.
2D Gel Electrophoresis: Provides higher resolution by first separating proteins based on pI and then size using SDS-PAGE.

M/S Process Overview: Measures molecular masses of ionized and fragmented molecules; separates ions based on mass-to-charge ratio (m/z).
- Data Output: Displays relative abundance of fragments vs m/z ratio, aiding in protein identification.
Identification Protocol:
1. Remove band/spot from the gel.
2. Digest protein with proteases (e.g., trypsin).
3. Separate resulting peptides via chromatography.
4. Analyze peaks with M/S for comparison against predicted peptide mass from digestion.

Use Cases of Gel Electrophoresis: Visualizes changes in protein expression across different conditions or treatments in various cell types.

Application Scope: Used to generate protein expression maps for various cell types under different conditions, identifying differential expressions based on molecular weight (Mw) and isoelectric point (pI).

Antibodies facilitate separation (immunoaffinity, immunoprecipitation) and identification (Western blot, immunofluorescence) of proteins.
- Monoclonal vs Polyclonal: Homogeneous population recognizing one epitope vs mixed population recognizing multiple epitopes.

Highly specific protein purification technique utilizing monoclonal antibodies against the target protein for efficient separation.

Utilizes enzyme- or luminescent-labelled antibodies for detection of specific proteins in a sample post-separation.

Uses fluorescently labelled antibodies to visualize structures (e.g., cytoskeleton) in cells using microscopy, enhancing the understanding of protein localization.