Protein Isolation
High-Yield Summary
- Two isolation technique families: electrophoresis (electric field) and chromatography (partitioning between stationary/mobile phases).
- Electrophoresis migration speed: v = Ez/f — velocity depends on field strength, net charge, and frictional coefficient (size/shape).
- Native PAGE preserves folded state (charge + shape); SDS-PAGE denatures + coats in negative SDS so separation is by size alone; isoelectric focusing separates by pI (protein stops moving once pH = pI, zero net charge).
- All chromatography relies on partitioning: more affinity for stationary phase = slower migration; more affinity for mobile phase = faster elution.
- TLC/paper chromatography separate by polarity, measured via Rf = distance moved by spot ÷ distance moved by solvent front.
- Column chromatography types: ion-exchange (charge), size-exclusion (size — small molecules enter pores and are SLOWED, large molecules bypass and elute FIRST), affinity (specific binding).
- GC separates vaporized compounds by retention time using an inert carrier gas; HPLC is a computer-controlled, high-precision version of column chromatography.
Key Terms
- Isoelectric point (pI)
- pH at which a protein carries zero net charge and stops migrating in an electric field.
- Partitioning
- Differential interaction of a compound with stationary vs. mobile phase — the basis of all chromatography separation.
- Rf value
- Retardation factor = distance moved by spot ÷ distance moved by solvent front (TLC/paper).
- Retention time
- Time for a compound to pass through a gas chromatography column; used to identify components.
Electrophoretic Migration Velocity
v = Ez / f
- v = Velocity of protein migration
- E = Electric field strength
- z = Net charge of the protein
- f = Frictional coefficient (depends on size/shape)
- Higher charge or field strength → faster migration; larger/bulkier protein (higher f) → slower migration.
Rf Value (TLC / Paper Chromatography)
Rf = distance moved by spot ÷ distance moved by solvent front
- Rf = Retardation factor, used to compare/identify compounds
- More nonpolar compounds travel farther (higher Rf); more polar compounds stick to the stationary phase (lower Rf).
Native PAGE vs. SDS-PAGE vs. Isoelectric Focusing
| Method | Separates By / Key Feature |
|---|---|
| Native PAGE | Size AND native charge/shape — folded state preserved, results harder to interpret |
| SDS-PAGE | Size ONLY — denatured, coated in negative SDS, original function not recoverable |
| Isoelectric focusing | Isoelectric point (pI) — stops at the pH where net charge = 0 |
Ion-Exchange vs. Size-Exclusion vs. Affinity Chromatography
| Type | Mechanism |
|---|---|
| Ion-exchange | Charged beads bind compounds of opposite charge |
| Size-exclusion | Porous beads: small molecules enter pores (slowed), large molecules bypass (elute first) |
| Affinity | Beads coated with a specific receptor/enzyme/antibody; target binds, rest washes out, then eluted |
TLC / Paper Chromatography Procedure
- 1Spot a small dot of sample near the bottom of the plate/paper.
- 2Place the bottom edge into solvent (mobile phase).
- 3Solvent rises by capillary action, carrying sample components at different rates based on polarity.
- 4Once the solvent front has traveled far enough, mark it and measure distance traveled by each spot.
- 5Calculate Rf for each spot to identify/compare compounds.
Common MCAT Trap
- Size-exclusion chromatography is counterintuitive: SMALL molecules elute LAST (they enter and get delayed in the pores), LARGE molecules elute FIRST (they bypass the pores).
- SDS-PAGE separation is by size alone, not charge — SDS masks the protein's native charge entirely, unlike Native PAGE.
- A protein at its pI has ZERO net charge and will NOT move under an electric field — don't assume more voltage will force migration at that pH.
Quick Recall
Why can't SDS-PAGE be used to study a protein's native function afterward?
What happens to a protein once it reaches its isoelectric point during isoelectric focusing?
In size-exclusion chromatography, which elutes first — large or small molecules?
What is the mobile phase in gas chromatography?