Unit 5: Analysis of proteins

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE), introduced by Ulrich Laemmli (1970), separates proteins purely by molecular mass by abolishing the influence of native charge and shape. It is the workhorse technique for checking protein purity, estimating subunit molecular weight, and comparing relative abundance across samples. The rest of this unit depends on the following governing ideas.

• Denaturation principle: SDS (an anionic detergent) unfolds proteins and coats them at a roughly constant ratio of ~1.4 g SDS per g protein, masking intrinsic charge so that charge-to-mass ratio becomes uniform.
• Sieving principle: The polyacrylamide matrix acts as a molecular sieve; migration rate through it depends on size, so smaller polypeptides move faster and travel farther.
• Reducing conditions: β-mercaptoethanol or dithiothreitol (DTT) cleaves disulphide (–S–S–) bonds, breaking multi-subunit proteins into individual polypeptide chains.
• Discontinuous buffer system: A low-percentage stacking gel (pH 6.8) concentrates proteins into a sharp band; a higher-percentage resolving gel (pH 8.8) separates them.
• Tracking and anchoring: Glycerol adds density so samples sink into wells; bromophenol blue marks the migrating dye front.
• Direction of migration: Because SDS-coated proteins are uniformly negative, all migrate toward the anode (+).

II. SDS-PAGE: Matrix, Reagents and Run

The physical and chemical system on which both analyses rest.

A. The polyacrylamide gel matrix

The separating medium is a cross-linked polymer whose pore size is tuned to the mass range of interest.

• Monomer and cross-linker: Acrylamide polymerises into chains; N,N′-methylenebisacrylamide cross-links them into a three-dimensional mesh.
• Polymerisation catalysts: Ammonium persulphate (APS) provides free radicals; TEMED (N,N,N′,N′-tetramethylethylenediamine) accelerates radical formation.
• Percentage governs pore size: Higher %T gives smaller pores for small proteins.
  • 7.5% gel: resolves ~50–200 kDa.
  • 10% gel: resolves ~20–100 kDa.
  • 12–15% gel: resolves ~10–40 kDa.
• Hazard note: Unpolymerised acrylamide is a neurotoxin and must be handled with gloves.

B. Sample and buffer chemistry

Sample treatment ensures every protein enters the gel in the same denatured, uniformly charged state.

• Laemmli sample buffer: contains Tris-HCl (pH 6.8), SDS, glycerol, β-mercaptoethanol and bromophenol blue.
• Heat treatment: samples boiled at 95–100 °C for ~5 min to complete unfolding and reduction.
• Running buffer: Tris-glycine-SDS at pH 8.3 supplies the mobile ions of the discontinuous system.
• Stacking mechanism: chloride (leading ion) and glycine (trailing ion) sandwich proteins into a thin zone before resolution begins.

C. Detection of separated bands

After the run, proteins are fixed and visualised in the gel.

• Coomassie Brilliant Blue R-250: binds protein non-specifically; detection limit ~50–100 ng per band.
• Silver staining: ~50–100× more sensitive (~1 ng), useful for trace proteins.
• Fixing and destaining: methanol–acetic acid fixes proteins; destaining removes background dye to reveal blue bands on a clear gel.

III. Qualitative analysis of protein using SDS PAGE

Establishing the identity, purity, number and molecular weight of polypeptides in a sample.

Qualitative analysis answers "what is present and how big is it," using band position and pattern rather than intensity.

A. Purity assessment

A single sharp band indicates a homogeneous preparation.

• Single band = pure: a purified protein shows one band at its expected mass.
• Multiple bands = impurity or subunits: extra bands signal contaminants, degradation products, or genuine subunits.
• Smearing: diffuse streaks indicate proteolytic degradation or overloading.

B. Determination of subunit composition

Reducing SDS-PAGE reveals the number and size of the chains that make up an oligomer.

• Oligomer dissociation: with DTT/β-ME, a tetramer resolves into its constituent monomers.
• Worked interpretation — haemoglobin: native ~64 kDa resolves into α (~16 kDa) and β (~16 kDa) chains, showing an α₂β₂ arrangement.
• Non-reducing comparison: running with and without reductant distinguishes disulphide-linked subunits from non-covalently associated ones.

C. Estimation of molecular weight

Molecular weight is read from a calibration curve of known standards.

• Relative mobility (Rf) is calculated for each band:

Rf = (distance migrated by protein) / (distance migrated by dye front)

• Standard curve: plot log₁₀(molecular weight) of marker proteins on the y-axis against Rf on the x-axis; the plot is approximately linear.
• Molecular weight markers: a protein ladder spanning e.g. 10, 15, 25, 35, 50, 70, 100, 130, 250 kDa.
• Reading the unknown: measure the unknown's Rf, locate it on the curve, and read off log MW.
• Worked example:
  • Dye front migrates 8.0 cm; unknown band migrates 4.0 cm → Rf = 0.50.
  • From the standard line, Rf 0.50 corresponds to log₁₀(MW) = 1.65 → MW ≈ 10^1.65 ≈ 45 kDa.
• Symbol key: Rf = relative mobility (dimensionless); MW = molecular weight in kDa.

D. Applications and limitations

• Applications: monitoring purification steps, checking recombinant protein expression, detecting proteolysis, confirming molecular weight.
• Limitations:
  • Charge and shape not measured: gives mass only, not native structure.
  • Glycoproteins run anomalously: attached carbohydrate makes them appear heavier than their true polypeptide mass.
  • Small peptides (<10 kDa) run off standard gels; require Tricine-SDS-PAGE.

IV. Quantitative analysis of protein using SDS PAGE

Measuring the relative or absolute amount of a protein from the intensity of its stained band.

Quantitative analysis rests on the principle that stain uptake is proportional to protein mass within a limited linear range.

A. Densitometry of stained bands

Band intensity is converted into a numerical value.

• Image capture: the destained gel is scanned or photographed under uniform illumination.
• Integrated optical density (IOD): software (e.g. ImageJ) measures the area × mean intensity of each band peak.
• Background subtraction: a blank region of the lane is subtracted to correct for stain haze.
• Linear range: Coomassie response is linear only over ~0.1–10 µg per band; above this, intensity saturates and underestimates amount.

B. Construction of a standard curve

Known amounts of a reference protein calibrate the band intensity.

• Loading series: run e.g. 0.5, 1, 2, 4, 8 µg of a standard protein such as BSA in adjacent lanes.
• Plot: band IOD (y-axis) against known mass (x-axis) yields a straight line through the linear region.
• Regression equation:

IOD = m × (protein mass) + c

• m = slope (IOD per µg); c = intercept.
  • Unknown quantitation: measure the sample band's IOD and solve for mass.
  • Worked example: standard line IOD = 1200·(µg) + 150; a sample band of IOD 3750 gives (3750 − 150)/1200 = 3.0 µg protein loaded.

C. Relative quantification and normalisation

Comparing a target across lanes requires correcting for loading differences.

• Loading control: a housekeeping protein band is quantified in every lane as a reference.
• Normalised value: target IOD ÷ loading-control IOD gives a ratio comparable between samples.
• Fold change: ratio in treated sample ÷ ratio in control reports up- or down-regulation.
• Percent purity estimate: (target band IOD ÷ total lane IOD) × 100 gives approximate purity of a preparation.

D. Sources of error and control

• Uneven staining/destaining: causes false intensity differences; process all lanes on one gel identically.
• Overloading: pushes bands out of the linear range, flattening quantitation.
• Gel-to-gel variation: standards and samples must be run on the same gel for valid comparison.
• Stain choice affects linearity: silver staining has a narrow, non-linear range and is poor for quantitation; Coomassie or fluorescent stains are preferred.

V. Practical Workflow Summary of the Technique

The ordered sequence linking preparation to result.

A. Step sequence

• Gel casting: pour resolving gel, overlay, then cast stacking gel with comb.
• Sample prep: mix with Laemmli buffer, boil 5 min.
• Loading: load ladder plus samples into wells.
• Electrophoresis: apply constant voltage (~100–200 V) until dye front reaches gel bottom.
• Staining: fix, stain with Coomassie, destain.
• Analysis: read band positions (qualitative) and band intensities (quantitative).

B. Critical control points

• Voltage and heat: excessive voltage overheats the gel, distorting bands ("smiling").
• Fresh APS/TEMED: stale catalysts give soft, incompletely polymerised gels.
• Consistent loading volume: equal total protein per lane underpins valid quantitative comparison.
• Marker choice: prestained markers allow visual tracking; unstained markers give sharper bands for accurate Rf measurement.