Adam Dawud

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3.3 - Techniques and Reagents

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Once we understand the properties of a protein, we can identify multiple different ways to sort proteins to figure out its composition.

Protein Analysis Techniques

SDS-PAGE

  • Principle: Separates proteins by size after denaturation with SDS.
  • Process:
    • SDS provides uniform negative charge.
    • Proteins move through a gel, with smaller proteins moving faster.
  • Application: Protein purity analysis and size determination.

Ion-Exchange Chromatography (IEC)

  • Principle: Separates proteins based on charge differences.
  • Process:
    • Resin with charged groups (cationic or anionic) binds proteins of opposite charge.
    • Elution by changing pH or salt concentration.
  • Application: Purifying proteins with distinct isoelectric points (pI).

Size-Exclusion Chromatography (SEC)

  • Principle: Separates based on size and shape.
  • Process:
    • Porous beads trap small proteins, larger proteins elute faster.
  • Application: Protein purification and molecular weight estimation.

Analytical Ultracentrifugation

  • Principle: Separates based on size and shape using centrifugal force.
  • Application: Determining protein complex formation and stoichiometry.

Mass Spectroscopy

  • Principle: Measures mass-to-charge ratio of ionized fragments.
  • Application: Identifying post-translational modifications and protein sequence.

Isoelectric Focusing (IEF)

  • Principle: Separates proteins based on their isoelectric point (pI).
  • Process:
    • Proteins migrate to the pH where their net charge is zero.
  • Application: Analyzing protein isoforms and charge variants.

Important Reagents

  • Iodoacetic Acid: Stabilizes cysteine by forming carboxymethyl cysteine.
  • Vinyl Pyridine: Forms pyridylethyl cysteine to prevent disulfide bond formation.
  • DTT & β-Mercaptoethanol: Break disulfide bonds (S-S) to free thiol groups (-SH).
  • Cyanogen Bromide (CNBr): Cleaves polypeptides at methionine residues.
  • Trypsin & Chymotrypsin: Cleave at specific residues (Lys/Arg and Phe/Tyr/Trp respectively).
  • Sodium Dodecyl Sulfate (SDS): Denatures proteins, providing a uniform negative charge.
  • 6N HCl: Hydrolyzes proteins into amino acids for analysis.