Asych Lecture 2-13.mp4

Recap of Column Chromatography

• Column chromatography fundamentals: involves a mobile phase (liquid) and a stationary phase (gel).

  • The mobile phase is pumped through the stationary phase that contains a gel.

Ion Exchange Chromatography

• Uses charged beads for separation:

  • DEAE cellulose: positively charged for anion exchange chromatography.

  • Phosphocellulose: negatively charged for cation exchange chromatography.

• Proteins' interaction with beads depends on their isoelectric point (pI).

  • High pI proteins (basic) stick to phosphocellulose; easily pass through DEAE.

  • Low pI proteins (acidic) stick to DEAE; easily pass through phosphocellulose.

• Elution scheme:

  • Fastest elution: similarly charged proteins.

  • Slowest elution: oppositely charged proteins.

  • Requires salt wash to outcompete ionic interactions in order to elute protein

Size Exclusion Chromatography

• Operates in contrast to gel electrophoresis:

  • In gel, smaller fragments move faster; in size exclusion, larger proteins elute first.

  • Analogy: small particles (glitter) stick to the column; larger particles separate out easily.

Affinity Chromatography

• Uses beads with attached molecules resembling protein substrates or agonists.

• The binding of proteins to beads is nearly irreversible, requiring denaturation methods (salt, pH, thermal) to elute.

• Most specific chromatography method due to its tailored binding approach.

Isoelectric Focusing (IEF)

• Similar to gel electrophoresis; separates proteins based on their pI.

  • Positively charged anode repels negatively charged proteins towards cathode and vice versa.

  • Proteins will stop migrating once they reach their pI, achieving net neutrality.

• Notable application:

  • Linus Pauling's study on sickle cell anemia using IEF to identify differences in hemoglobin.

Sickle Cell Anemia and IEF

• Sickle cell mutation:

  • A single nucleotide change (glutamic acid to valine) alters hemoglobin properties.

  • Identified distinct bands in IEF: normal hemoglobin (one band), sickle cell (two bands).

Gel Electrophoresis and SDS-PAGE

• Traditional method for analyzing proteins based on size.

• SDS-PAGE:

  • Treats proteins with SDS to give negative charge, allowing separation solely by size.

  • Larger proteins travel less far than smaller proteins.

• Two-Dimensional (2D) Gel Electrophoresis:

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

Western Blotting

• Developed for identifying specific proteins from gels.

• Process involves:

  1. Transfer proteins to a membrane after SDS-PAGE.

  2. Blocking with skim milk to prevent non-specific binding.

  3. Binding with primary and secondary antibodies:

     • Secondary antibodies are enzyme-conjugated for chemiluminescent detection.

• Critical for determining protein expression levels, requiring control (housekeeping proteins) for validation.

  • Common housekeeping genes: Tubulin, Actin.

Techniques for Protein Identification

Mass Spectrometry

• Ionizes and separates molecules; calculates time of flight to determine mass/charge ratios.

• Useful for peptide sequences and metabolite identification.

X-ray Crystallography

• Purifies protein, crystallizes it, then uses X-ray beams to discern structure.

• Limitations: long process, difficult to crystallize all proteins, potential resolution issues.

Cryo-Electron Microscopy (Cryo-EM)

• Recent development for rapid structural analysis of proteins.

• Uses grid for sample preparation, collecting various 2D images for 3D reconstruction.

• Advantage: Faster than crystallography, useful for flexible protein regions.

AlphaFold

• Google DeepMind algorithm predicts protein structures from sequences, revolutionizing structural biology.

• Eliminates much guesswork in protein synthesis design.

Closing Remarks

• Supplementary Material: Recommended Veritasium video on AlphaFold to further understanding.

• Encouragement to engage with materials over the weekend before the next class.