Protein Analysis Methods

Protein Basics and Structure

  • Amino acid sequence determines protein function.
  • Proteins have primary, secondary, tertiary, and quaternary structures.
  • Protein folding dictates functional form.

Protein Purification and Isolation

  • Centrifugation: Separates proteins by mass and density.
  • Precipitation: Uses ammonium sulfate to isolate proteins.
  • Chromatography Techniques:
    • Ion exchange: Separates by charge.
    • Gel filtration: Separates by size.
    • Affinity: Uses specific binding.

Quantitative and Qualitative Analysis

  • SDS-PAGE: Separates proteins by size.
  • Isoelectric Focusing (IEF): Separates by isoelectric point.
  • Western Blotting: Uses antibodies to detect specific proteins.
    • Monoclonal and Polyclonal

Protein Characterization

  • Sequence Analysis: Determines amino acid sequence.
    • Hydrolysis and Edman degradation
  • Structure Elucidation:
    • X-ray crystallography: High-resolution 3D structures.
    • NMR: For soluble proteins, studies dynamics.
    • Cryo-EM: For large complexes.
    • Mass spectrometry (MS): Identifies masses, PTMs, interactions.

Protein Aufreinigung and Isolierung

  • Cell Lysis Methods:
    • Mechanical: Ultraschall (Sonication), French-Press-Verfahren
    • Chemical: Detergents, Osmotic shock
    • Enzymatic: Lysozym, Proteases
    • Thermische: Gefrier-Tau-Zyklen, Erhitzen
  • Vorreinigung:
    • Zentrifugation: Niedriggeschwindigkeits-, Ultrazentrifugation
    • Filtration: Großporige, Feinfiltration (Sterilfiltration)
  • Dichtegradientenzentrifugation: Separates particles by density.
    • High resolution, protection of proteins
  • Fällung (Precipitation):
    • Aussalzung with Ammoniumsulfat
      • Konzentrieren verdünnter Proteinlösungen
    • Organische Lösungsmittel
      • Fällen with kaltem Aceton oder Ethanol(Denaturierung)
      • Fällen with Trichloressigsäure (TCA-Fällung)(Denaturierung)
  • Dialyse: Separates molecules by size using a membrane.
    • Ideal zum entfernen hoher Salzkonzentrationen

Chromatographie

  • Separates substances based on interactions between mobile and stationary phases.
  • Techniques:
    • Gelfiltration
    • Adsorption
    • Verteilung
    • lonenaustausch
    • Affinität

Gelfiltration

  • Separates by molecule size using porous material (SEC).
    Molecule >> Pore: Eddy diffusion;
    Molecule << Pore: Eddy diffusion;

Adsorption Chromatographie

  • Separates based on polarity using materials like Kieselgel.

Verteilung Chromatographie

  • Separates based on polarity using immiscible phases.
  • Hydrophile Interaktions Chromatographie (HILIC)

Ionenaustausch Chromatographie

  • Separates by charge using ionic interactions with a matrix.

Affinität Chromatographie

  • Uses specific binding pairs.
  • Ligands: Antikörper ↔ Antigen
  • Mono-specific ligands, Group-specific ligands

HPLC/UPLC

  • High-performance liquid chromatography for improved separation using pressure.

Elektrophorese

  • Separates molecules in an electric field by size, charge, and structure.
  • Applications:
    • SDS-PAGE
    • Native PAGE
    • Isoelektrische Fokussierung
    • 2D-Gelektrophorese

(Dis)-kontinuierliche Elektrophorese

  • Uses stacking and resolving gels for better separation.

Native Polyacrylamid Gel-Elektrophorese

  • Separates proteins in native form by size and charge.

Sodium Dodecyl Sulfate – PAGE

  • SDS PAGE is a Methode zur Trennung von Proteinen basierend ihrer Größe unter denaturierenden Bedingungen
  • Proteins are treated with SDS, denaturing them and providing a negative charge proportional to size.

Isoelektrische Fokusierung

  • Separates proteins based on isoelectric point (pI) in a pH gradient.

2D-Gelektrophorese

  • Combines IEF and SDS-PAGE for high-resolution protein separation.

Visualisierung

  • Coomassie-Brilliant-Blau
  • Fluoreszenzfärbung
  • Silberfärbung

Indirekte Visualisierung

  • Western Blot: Protein

Quantifizierungs- Methoden

  • Chemische Quantifizierung
  • Physikalische Quantifizierung
  • Biochemische Quantifizierung

Chemische Quantifizierung

  • Ninhydrin Assay: Measures free amines.
  • Biuret Assay: Measures peptide bonds.
  • Bicinchoninsäure Assay: Hohe Sensitivität
  • Bradford Assay: Uses dye binding.
  • Sehr hohe Sensitivität

Physikalische Quantifizierung

  • UV/Vis Spektroskopie:
    • Uses UV/Vis absorption to measure concentration.
    • Nukleinsäuren, Proteine

Biochemische Quantifizierung

  • Polyklonale Antikörper
  • ELISA:

Aminosäuresequenzen – Hydrolyse / Edman Abbau