Protein Separation Techniques — 1st Year MBBS

Overview

I. PRECIPITATION METHODS

1. Salting Out (most commonly asked)

• Principle: Differential solubility based on salt concentration
• High concentrations of ammonium sulfate (NH₄)₂SO₄ remove the water of hydration around proteins, reducing solubility → proteins precipitate
• Albumin stays in solution at high (NH₄)₂SO₄ concentrations that precipitate globulins
• Used clinically: Cohn's ethanol fractionation isolates immunoglobulins, albumin (fraction V), α- and β-globulins from plasma

2. Isoelectric Precipitation

• Proteins are least soluble at their isoelectric point (pI)
• Adjusting pH to pI → protein precipitates

3. Organic Solvent Precipitation

• Addition of ethanol or acetone reduces polarity of solvent → proteins precipitate

II. ELECTROPHORESIS

A. Serum Protein Electrophoresis (SPE) — most clinically important

• Principle: Proteins migrate in an electric field based on their charge and size
• At pH 8.6, proteins separate into bands:
  • Albumin (fastest, most negative, toward anode)
  • α₁-globulin
  • α₂-globulin
  • β-globulin
  • γ-globulin (slowest, least negative / toward cathode)
• Support media: cellulose acetate, agarose gel, starch gel, polyacrylamide
• Stains used: Coomassie Brilliant Blue, Ponceau S, Amido Black; Silver stain for trace proteins; Oil Red O / Sudan Black for lipoproteins

Electroosmosis (Endosmosis): When the support medium has a negative charge, buffer flows toward the cathode, carrying proteins with it.

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

• SDS (anionic detergent) denatures proteins and gives all polypeptides a uniform negative charge proportional to mass
• Separation is purely by molecular size (molecular weight)
• β-mercaptoethanol / dithiothreitol used to reduce disulfide bonds
• Bands visualized by Coomassie Blue staining
• Principle: Larger proteins → more resistance → migrate less

C. Isoelectric Focusing (IEF)

• A pH gradient is established in polyacrylamide gel using ampholytes
• Proteins migrate until they reach the pH = their isoelectric point (pI), where net charge = 0 → they stop (focus)
• Separates proteins based on charge (pI) alone

D. Two-Dimensional (2D) Electrophoresis

• 1st dimension: IEF (separates by pI / charge)
• 2nd dimension: SDS-PAGE (separates by molecular weight)
• Extremely high resolution — separates complex protein mixtures (used in proteomics)

E. Capillary Zone Electrophoresis (CZE)

• Separation in a narrow capillary tube with high-resolution separation
• More advantageous than agarose or cellulose acetate for serum protein analysis

III. CHROMATOGRAPHY

IV. ULTRACENTRIFUGATION

• Separates proteins based on size, shape, and density using centrifugal force
• Sedimentation coefficient (S) used to characterize proteins
• Used for: ribosomes, lipoproteins, large protein complexes

V. DIALYSIS

• Not a separation technique per se, but used for purification
• Uses a semipermeable membrane to remove small molecules (salts, small metabolites) while retaining large proteins
• Used to remove ammonium sulfate after salting out

Quick Exam Summary Table

Most Exam-Relevant Points to Remember

1. Tiselius introduced moving boundary electrophoresis — first described 4 serum fractions
2. pH 8.6 is used for serum protein electrophoresis (albumin is most negative → runs fastest toward anode)
3. SDS-PAGE separates by size only (SDS masks all charges)
4. IEF separates by charge (pI) only
5. Gel filtration — large molecules elute first (excluded from pores)
6. Affinity chromatography is the most specific method
7. 2D electrophoresis = IEF + SDS-PAGE = used in proteomics
8. Silver staining is most sensitive for detecting trace proteins on gels