Genomics and Proteomics (1-2)

What is SDS-PAGE?

• Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis.

• A laboratory technique used to separate proteins based on their molecular weight.

Chemical properties of SDS

• Structure:

  • Long hydrophobic alkyl chain (dodecyl, 12 carbons).

  • Negatively charged sulfate head group (–OSO₃⁻) with sodium counterion (Na⁺).

• Type: Anionic surfactant/detergent.

• Amphipathic nature

  • Hydrophobic tail + hydrophilic sulfate head.

  • Allows SDS to dissolve hydrophobic molecules in water (detergent property).

• Ionic character

  • Sulfate group makes SDS strongly negatively charged in aqueous solution.

  • This is what gives proteins a uniform negative charge during SDS-PAGE.

• Denaturing ability

  • SDS disrupts non-covalent bonds (hydrogen bonds, hydrophobic interactions).

  • This unfolds (denatures) proteins, giving them a rod-like shape.

What does SDS do to proteins?

Before SDS

• The protein is in its native folded state.

• Structure stabilized by:

  • Hydrophobic interactions (H in the diagram = hydrophobic patches tucked inside).

  • Charged R-groups (+ and –) interacting on the surface.

• Result: A complex 3D shape, and each protein has its own unique net charge (depending on amino acids).

After SDS

• SDS (a strong anionic detergent) binds along the length of the protein.

• It:

  1. Denatures the protein → unfolds it into a linear chain.

  2. Masks natural charges → coats the protein with negative charges (–).

• Now, all proteins have:

  • Similar rod-like shapes.

  • A uniform negative charge proportional to their length.

How much SDS binds to protein?

About 1.4 g SDS per 1 g of protein, binding uniformly along hydrophobic regions.

What happens if a cell is incubated with SDS?

Membranes dissolve, all proteins are solubilized, and all proteins are coated with many negative charges.

If proteins are denatured with SDS and placed in just an electric field, what happens?

They all move toward the positive pole at the same rate → no size-based separation.

What medium is used to separate proteins of different sizes?

Polyacrylamide gel, which slows large proteins more than small ones.

What is the name of the process combining SDS and polyacrylamide gel?

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

Why do small proteins move faster in SDS-PAGE?

Because polyacrylamide gel acts like a molecular sieve → small molecules move more easily through pores.

Where do large proteins stay in SDS-PAGE?

Closer to the well (top of the gel), since they move more slowly.

What are the two important outcomes of SDS-PAGE?

• Proteins lose all structure beyond primary structure.

• All proteins have a large negative charge and migrate toward the positive pole.

PTM crosstalk: Biology

• Shows how PTMs occur in biological systems.

• PTMs can:

  • Be added at specific amino acids (red/blue dots).

  • Occur in patterns or multiple sites along a protein.

  • Cause folding/unfolding changes or regulate protein-protein interactions.

PTM crosstalk: proteomic methods

• Methods to detect PTMs using mass spectrometry (MS).

• Workflow: proteins are digested, PTMs are mapped, then peptides are analyzed by MS.

• Specialized MS strategies allow detection of different modifications.

PTM crosstalk:

• PTMs affect protein function, stability, localization, and interactions.

• Experimental strategies:

  • Knockdown/knockout of modified proteins.

  • Enrichment of modified peptides.

  • MS-based functional analysis.