Unit 6: Detection of proteins by western blotting

Western blotting is an immunochemical technique that transfers electrophoretically separated proteins onto a membrane and identifies a specific target using antibodies. Developed by W. Neal Burnette (1981), who coined the name as a pun on Southern blotting (DNA) and northern blotting (RNA), it combines the resolving power of gel electrophoresis with the specificity of antigen–antibody recognition.

• Purpose: Confirm the presence, relative size and approximate amount of a chosen protein in a complex mixture such as a cell lysate.
• Three-stage logic: Separate by molecular weight → Transfer to a solid support → Probe with antibodies and visualise.
• Specificity source: The primary antibody binds a single epitope, so a single band appears against many co-migrating proteins.
• Semi-quantitative: Band intensity is proportional to antigen amount only within a limited linear range; a loading control (e.g. β-actin, GAPDH) normalises comparisons.
• Key reagents recurring below: SDS, polyacrylamide, PVDF or nitrocellulose membrane, blocking agent (BSA / non-fat milk), primary and secondary antibodies, enzyme substrate.

II. Sample Preparation and SDS-PAGE

Denaturation and size-based separation of the protein mixture.

A. Principle and purpose

Proteins are denatured to linear, uniformly charged chains so that electrophoretic mobility depends only on molecular weight.

• SDS action: Sodium dodecyl sulphate coats the polypeptide backbone at roughly one molecule per two amino-acid residues, masking intrinsic charge and imposing a constant negative charge-to-mass ratio.
• Reducing agent: β-mercaptoethanol or dithiothreitol (DTT) cleaves disulphide (–S–S–) bonds so subunits migrate independently.
• Heat step: Boiling at 95–100 °C for ~5 min completes unfolding.

B. Gel and run conditions

The polyacrylamide matrix acts as a molecular sieve.

• Discontinuous system: A wide-pore stacking gel (~4 %) concentrates samples into sharp bands; the resolving gel (8–15 %) separates them.
• Pore size vs. size range: Higher acrylamide % resolves small proteins; lower % resolves large ones.
• Mobility relation: Migration distance is linear with the logarithm of molecular weight.

log(MW) = -m·(Rf) + c
Rf = distance migrated by protein / distance migrated by dye front

• Symbols: MW = molecular weight (Da); Rf = relative mobility; m = slope; c = intercept from marker calibration.
• Marker ladder: Pre-stained standards (e.g. 10–250 kDa) allow size estimation of the target band.

III. Protein Transfer (Blotting)

Moving separated proteins from gel to an immobilising membrane.

A. Principle and membranes

Proteins are eluted from the gel and immobilised on a membrane that presents them for antibody access.

• Driving force: An electric field pulls negatively charged SDS–protein complexes from gel (cathode side) toward the membrane (anode side).
• Nitrocellulose: Binds protein by hydrophobic interaction; good for low-background chemiluminescence.
• PVDF: Polyvinylidene difluoride; higher protein-binding capacity and mechanical strength, requires methanol pre-wetting.

B. Transfer methods

Two arrangements move protein out of the gel.

1. Wet (tank) transfer: Gel-membrane sandwich fully submerged in cold buffer; slow (1 h–overnight) but complete, preferred for large proteins.
2. Semi-dry transfer: Sandwich pressed between buffer-soaked plate electrodes; fast (15–30 min), lower buffer volume, better for small-to-mid proteins.

• Transfer buffer: Tris-glycine with 20 % methanol; methanol strips SDS and improves membrane binding but slows elution of large proteins.
• Verification: Ponceau S stain reversibly marks total transferred protein and confirms even transfer before probing.

IV. Blocking and Immunodetection

Preventing non-specific binding, then targeting the antigen with antibodies.

A. Blocking

Unoccupied membrane sites are saturated so antibodies bind only their antigen.

• Blocking agents: 5 % non-fat dry milk or 3–5 % bovine serum albumin (BSA) in TBS-T; BSA is chosen for phospho-protein studies because milk casein is a phosphoprotein that raises background.
• Detergent: 0.05–0.1 % Tween-20 in wash buffer reduces weak hydrophobic sticking.

B. Primary and secondary antibodies

Detection uses a two-antibody sandwich for amplification.

1. Primary antibody: Binds directly and specifically to the target epitope; monoclonal (single epitope, high specificity) or polyclonal (multiple epitopes, higher sensitivity).
2. Secondary antibody: Anti-species IgG carrying the reporter enzyme; several secondaries bind one primary, amplifying signal.

• Enzyme conjugates: Horseradish peroxidase (HRP) or alkaline phosphatase (ALP) is covalently linked to the secondary antibody.
• Wash steps: Repeated TBS-T washes between incubations remove unbound antibody and lower background.

V. Detection of Proteins by Western Blotting

Converting the antibody–enzyme complex into a visible, recordable signal.

A. Detection chemistry

The membrane-bound enzyme catalyses a substrate reaction localised at the antigen band.

• Chemiluminescence (ECL): HRP oxidises luminol in the presence of H₂O₂ and an enhancer, emitting light captured on X-ray film or a digital imager; most common, highly sensitive (picogram range).

Luminol + H2O2  --HRP-->  3-aminophthalate + light (~425 nm)

• Colorimetric: HRP with DAB, or ALP with BCIP/NBT, deposits an insoluble coloured precipitate at the band; permanent record but lower sensitivity.
• Fluorescence: Fluorophore-tagged secondary antibodies emit light on excitation, enabling multiplexing of several targets by different colours on one membrane.

B. Result interpretation

The output is read for identity, size and amount.

• Band position: Compared against the ladder to confirm the expected molecular weight of the target.
• Band intensity: Densitometry gives relative abundance; values are normalised to the loading control.
• Loading control: A housekeeping protein of constant expression verifies equal sample loading across lanes.

Normalised signal = target band density / loading-control band density

• Symbols: density values are integrated pixel intensities from imaging software (arbitrary units).

C. Common artefacts and troubleshooting

Signal defects trace back to specific stages.

• High background: Insufficient blocking, too much antibody, or inadequate washing.
• No signal: Failed transfer, degraded antibody, or absent antigen; Ponceau check distinguishes transfer failure.
• Multiple bands: Protein degradation, non-specific antibody, or post-translational modification (e.g. glycosylation shifting apparent mass).
• "Smiling" bands: Overheating during electrophoresis; run at lower voltage or cool the tank.

D. Applications and limitations

The technique answers "is this specific protein present, and how much?".

• Diagnostics: Confirmatory HIV testing detects anti-viral antibodies; also used for Lyme disease and BSE screening.
• Research uses: Verifying protein expression after cloning, checking knockdown efficiency, and detecting post-translational modifications with phospho-specific antibodies.
• Sensitivity: Detects nanogram to picogram amounts of target protein.
• Limitations: Depends on antibody quality and availability; only semi-quantitative; time-consuming (often two days); denaturation destroys conformational epitopes so some antibodies fail.

VI. Comparison with Related Blotting Techniques

Placing the method among the transfer-and-probe family.

A. The blotting family contrasted

All three transfer separated biomolecules to a membrane, differing in target and probe.

1. Southern and northern blotting: Detect DNA (Southern) and RNA (northern) using labelled nucleic-acid probes that hybridise by base pairing.
2. Western blotting: Detects protein using antibody probes that recognise epitopes, not sequence complementarity.

• Shared workflow: Electrophoretic separation → membrane transfer → specific probe → signal detection.
• Distinguishing feature: Only western blotting relies on antigen–antibody affinity, giving it access to protein-level information such as size, abundance and modification state that nucleic-acid blots cannot provide.

B. Position among protein assays

Western blotting complements other protein methods.

• Versus ELISA: ELISA quantifies antigen faster and in plates but gives no size information; western blotting adds the molecular-weight dimension, distinguishing the true target from cross-reacting species.
• Versus mass spectrometry: MS identifies proteins de novo without antibodies; western blotting is cheaper and antibody-directed, ideal when the target is already known.
• Combined use: A protein first identified by MS is routinely confirmed and monitored across samples by western blotting, making the two complementary rather than competing.