🧬 Protein Separation Techniques

Short Notes — Based on Vasudevan's Textbook of Biochemistry for Medical Students

OVERVIEW FLOWCHART

PROTEIN MIXTURE (e.g., serum, tissue extract)
              │
              ▼
    ┌─────────────────────┐
    │  STEP 1: Extraction │ ← Homogenization / solubilization
    └─────────┬───────────┘
              │
              ▼
    ┌─────────────────────────────────┐
    │  STEP 2: Preliminary Separation │
    │  • Salting Out                  │
    │  • Dialysis                     │
    │  • Centrifugation               │
    └─────────┬───────────────────────┘
              │
              ▼
    ┌─────────────────────────────────┐
    │  STEP 3: Fine Purification      │
    │  • Chromatography               │
    │  • Electrophoresis              │
    │  • Ultracentrifugation          │
    └─────────┬───────────────────────┘
              │
              ▼
       PURE PROTEIN (checked by PAGE / IEF)

1. ELECTROPHORESIS

Principle: Movement of charged particles in an electric field.

• Cations (positive) → move to cathode (−)
• Anions (negative) → move to anode (+)
• Proteins carry charge depending on pH → separate by charge + size + shape

Factors Affecting Migration Rate

NET CHARGE (pI of protein)
        +
  pH OF MEDIUM
        +
 MASS & SHAPE
        +
 FIELD STRENGTH
        +
SUPPORT MEDIUM
        +
  TEMPERATURE
       ──────────────────────
       All determine SPEED OF MIGRATION

Types of Electrophoresis

               ELECTROPHORESIS
                     │
     ┌───────────────┼────────────────────┐
     ▼               ▼                    ▼
Paper (16–18h)   Cellulose Acetate     Agarose Gel
Low voltage      (1 hour, better       Serum proteins,
Diffusion        resolution)           nucleic acids
problem          Used for lipoproteins  90 min run
     │               │
     ▼               ▼
           ┌─────────────────┐
           │ PAGE (Polyacryl- │  → Separates by size
           │ amide Gel)       │  → SDS-PAGE: all proteins
           │                  │    negatively charged
           └─────────────────┘
                  │
                  ▼
        ┌─────────────────────┐
        │ Isoelectric Focusing│  → Separates by pI only
        │ (IEF)               │  → Sample in nanogram range
        └─────────────────────┘
                  │
                  ▼
        ┌──────────────────────────┐
        │ High Voltage Electrophor.│ 400–2,000 V → result
        │ (HVE)                    │ in <30 minutes
        └──────────────────────────┘
                  │
                  ▼
        ┌──────────────────────────┐
        │ Capillary Electrophoresis│ 25,000 V, nanoliter
        │                          │ sample, few minutes
        └──────────────────────────┘

Serum Protein Bands on Electrophoresis

(−) Cathode ←──────────────────────── Anode (+)
                                           ↑
    γ    β    α2   α1   Albumin  ←── direction of migration
(slowest)                       (fastest, most negative)

2. CHROMATOGRAPHY

Principle: Separation of proteins based on differential interaction with a stationary phase as they move through it with a mobile phase.

Master Flowchart of Chromatography Types

            CHROMATOGRAPHY
                  │
    ┌─────────────┼──────────────┐
    ▼             ▼              ▼
ADSORPTION    PARTITION      ION-EXCHANGE
(Alumina /    (Paper /        (Separates by
 Silica gel)   TLC / GLC)      charge)
    │
    │
    ▼
GEL FILTRATION        AFFINITY
(Size Exclusion)      (Specific binding)

A. Adsorption Chromatography

Protein mixture applied on column
        │ (alumina or silica gel packed)
        ▼
Weakly adsorbed proteins
        │ → elute first (move fastest)
        ▼
Strongly adsorbed proteins
        │ → elute last (move slowest)
        ▼
Fractions collected → analyzed

B. Ion-Exchange Chromatography

CATION EXCHANGE RESIN        ANION EXCHANGE RESIN
(Negatively charged resin)   (Positively charged resin)
        │                            │
Binds (+) charged proteins    Binds (−) charged proteins
        │                            │
Elute with increasing         Elute with increasing
salt concentration            salt concentration

C. Gel Filtration (Size Exclusion) Chromatography

Protein mixture added to top of column
(Sephadex beads with pores)
          │
          ▼
LARGE proteins → CANNOT enter beads
          → travel around beads
          → EXIT FIRST (elute faster)
          │
SMALL proteins → ENTER beads
          → take longer path
          → EXIT LAST (elute slower)
          │
          ▼
Fractions collected → separated by molecular size

D. Affinity Chromatography

Column contains SPECIFIC LIGAND
(e.g., NAD for dehydrogenases,
 Antigen for antibodies)
          │
Protein mixture added
          │
          ▼
SPECIFIC protein binds to ligand → RETAINED
OTHER proteins → pass through → washed away
          │
          ▼
Bound protein ELUTED by:
• Changing pH / salt
• Competing ligand
          │
          ▼
HIGHLY PURE protein recovered

E. HPLC (High Performance Liquid Chromatography)

Same principles as above BUT:
• Uses high pressure pumps
• Very fine particles in column
• Automated detection
• Result in MINUTES
• Very high resolution + sensitivity

3. PRECIPITATION REACTIONS (Preliminary Purification)

Salting Out

Neutral salt (NH4)2SO4 or Na2SO4 added
          │
          ▼
Salt ions compete for water molecules
          │
          ▼
Shell of hydration REMOVED from protein
          │
          ▼
Protein-protein interactions increase
          │
          ▼
PROTEIN PRECIPITATES
          │
NOTE: Higher molecular weight protein → precipitates at LOWER salt concentration

Dialysis

Protein solution placed in
semi-permeable membrane (dialysis bag)
          │
Immersed in large volume of buffer
          │
          ▼
SMALL molecules (salts, small metabolites)
→ diffuse OUT through membrane pores
          │
LARGE proteins
→ RETAINED inside bag
          │
          ▼
Salt-free, purified protein solution

4. ULTRACENTRIFUGATION

Principle: Molecules sediment at different rates based on size, shape, and density when spun at very high speeds (up to 500,000 × g).

PROTEIN MIXTURE in centrifuge tube
          │
HIGH-SPEED SPINNING (ultracentrifuge)
          │
          ▼
LARGE, DENSE particles → sediment FAST
          │                (high S value)
SMALL, LIGHT particles → sediment SLOW
          │                (low S value)
          ▼
Sedimentation coefficient (S) measured
        (Svedberg units)
          │
          ▼
Molecular weight calculated

• Ribosome 80S (eukaryote) = 60S + 40S subunits
• Ribosome 70S (prokaryote) = 50S + 30S subunits

5. IMMUNOELECTROPHORESIS

Step 1: Electrophoresis of serum proteins in agarose gel
          │
          ▼
Step 2: Antiserum placed in a trough cut parallel to the
        separated protein bands
          │
          ▼
Step 3: Antibodies and antigens DIFFUSE toward each other
          │
          ▼
Step 4: Where they meet at equivalence →
        PRECIPITATION ARCS (precipitin lines) form
          │
          ▼
Qualitative identification of proteins
(e.g., myeloma proteins, Bence-Jones proteins)

6. COMPARISON SUMMARY TABLE

7. KEY POINTS TO REMEMBER (Exam Focus)

┌─────────────────────────────────────────────────────────┐
│ • Tiselius introduced electrophoresis → Nobel Prize 1948│
│ • At pH 8.6, serum separates into:                      │
│   Albumin > α1 > α2 > β > γ (anode to cathode)         │
│ • Affinity chromatography = most specific & powerful    │
│ • Gel filtration: BIG exits FIRST                       │
│ • Ion exchange: proteins bind based on CHARGE           │
│ • SDS-PAGE: all proteins negative → separate by SIZE    │
│ • Salting out: higher MW protein precipitates FIRST     │
│ • Dialysis removes SMALL molecules, retains proteins    │
│ • Svedberg (S) unit = measure of ultracentrifugation    │
│ • Immunoelectrophoresis: combines electrophoresis +     │
│   immunodiffusion (Ouchterlony technique)               │
└─────────────────────────────────────────────────────────┘