Protein Detection Techniques – Western Blotting, ELISA & Multiplex Assays

Learning Objectives

• Ability to describe the principles and generic steps of:

  • Polyacrylamide Gel Electrophoresis (PAGE) & Western blotting

  • Enzyme-Linked Immunosorbent Assay (ELISA)

  • Multiplex immunoassay (Luminex®)

• Capacity to discuss differences among these techniques and decide which is most appropriate for a specific experimental design.

Polyacrylamide Gel Electrophoresis (PAGE)

• Two main formats

  • Native PAGE

  • Separates proteins in their native (non-denatured) state.

  • Separation based on combined influence of:

    • Charge density (charge-to-mass ratio)

    • Size

    • Shape

  • Conducted at 4^\circ C to preserve protein conformation and activity.

  • No pre-treatment with heat, SDS, or other denaturants → proteins retain biological function for downstream assays (e.g., activity tests, binding studies).

  • SDS-PAGE (Sodium Dodecyl Sulphate – PAGE)

  • SDS binds along the polypeptide chain (roughly 1 SDS per 2 amino acids).

  • Confers uniform negative charge → electrophoretic mobility depends mainly on molecular weight.

  • Sample preparation may include reducing agents (e.g., \beta-mercaptoethanol, dithiothreitol) to break disulphide bonds and fully linearise protein.

  • Produces straight molecular-weight ladder of bands (reference markers used).

Western Blotting (Immunoblotting)

• Principle: Combines PAGE separation with antibody-based detection on a membrane.

  1. Separation of proteins in polyacrylamide gel (usually SDS-PAGE).

  2. Electro-transfer of resolved proteins onto solid membrane (nitrocellulose or PVDF).

  3. Immunodetection of specific target(s) with antibodies.

• Transfer methods

  • Semi-dry transfer

  • Wet tank transfer (classical; higher buffer volume)

  • Goal: move proteins out of gel onto membrane using electric current, preserving band pattern.

• Blocking

  • Membrane pores not occupied by protein are filled with irrelevant protein (e.g., 5\% skim milk in PBS, 1\% BSA) to prevent non-specific antibody binding.

• Antibody probing sequence

  1. Incubate with primary antibody (target-specific).

  2. Wash thoroughly to remove unbound primary Ab.

  3. Incubate with secondary antibody (species-specific, enzyme-conjugated; typically HRP or AP).

  4. Wash again.

  5. Detect signal (chemiluminescence widely used; alternatives: chromogenic, fluorescent).

• Normalization & Semi-Quantification

  • Signal intensity of target band measured by densitometry.

  • Normalized to housekeeping protein (GAPDH, \beta-actin, tubulin, etc.) to account for loading/transfer variability.

  • Relative expression = \frac{\text{Target band densitometry}}{\text{Housekeeper densitometry}}.

Western Blot Advantages / Disadvantages (expanded from homework)

• Advantages

  • High specificity via two antibodies.

  • Provides molecular weight confirmation (band position).

  • Allows detection of post-translational modifications (using PTM-specific antibodies).

  • Semi-quantitative; multiple proteins can be probed sequentially (stripping/re-probing).

• Disadvantages

  • Labor-intensive, multi-step; time-consuming.

  • Limited throughput; generally one or few proteins per blot.

  • Semi-quantitative, not fully quantitative without rigorous standards.

  • Requires relatively large protein amounts compared with ELISA.

When to Use Western Blot (conceptual guide)

• Sample: cell lysates, tissue homogenates, sub-cellular fractions where protein is abundant enough for blotting.

• Target location: intracellular, membrane, nuclear proteins where size verification is valuable.

• Suitable for confirming expression, size variants, cleavage products, or PTMs after gene manipulation or treatment.

Enzyme-Linked Immunosorbent Assay (ELISA)

• Developed 1971 (Engvall & Perlmann). Non-radioactive successor to Radioimmunoassay (RIA).

• Core concept: Immobilise antigen or antibody on a solid phase (microplate well) → probe with enzyme-labelled antibody → enzymatic reaction generates measurable signal proportional to analyte quantity.

• Advantages vs RIA

  • No radioactivity → safer, cheaper disposal.

  • Similar sensitivity; amenable to automation.

ELISA Detection Chemistries

1. Chemiluminescent (most sensitive; transient light emission).

2. Chemifluorescent (light emitted upon electron relaxation; detect in fluorimeter).

3. Colorimetric (most common in research).

   • Example substrate: TMB (3,3',5,5'-tetramethylbenzidine); read optical density at \approx 450\,\text{nm} after stop solution.

Generic ELISA Workflow

1. Coating / Capture

   • Direct adsorption of antigen OR pre-coating with capture antibody.

2. Blocking

   • Add irrelevant proteins (e.g., BSA, casein) to occupy remaining binding sites.

3. Probing / Detection

   • Incubate with antigen-specific antibody (primary), possibly followed by enzyme-labelled secondary antibody.

4. Signal Measurement

   • Add substrate → measure absorbance / luminescence / fluorescence.

5. Extensive washing between steps 1–3 critical to minimise false positives.

ELISA Formats

• Direct ELISA

  • Labelled primary antibody binds directly to antigen.

  • Simple, quick; higher background due to limited signal amplification.

• Indirect ELISA

  • Unlabelled primary Ab + enzyme-linked secondary Ab.

  • Amplifies signal; more flexible (same secondary for many primaries).

• Sandwich / Capture ELISA

  • Two antibodies recognise distinct epitopes → “sandwiches” antigen.

  • Highest specificity & sensitivity; can measure complex samples (serum, plasma).

  • Variants: direct sandwich (labelled detection Ab) or indirect sandwich (secondary Ab).

• Competitive ELISA

  • Useful for small antigens with single epitope (e.g., hormones).

  • Signal inversely proportional to antigen concentration.

Data Interpretation

• Quantitative (primary application): generate standard curve with known antigen dilutions → interpolate sample concentrations.

• Semi-Quantitative: compare optical densities among samples without absolute units.

• Qualitative: yes/no presence above blank.

ELISA Advantages / Disadvantages (homework elaborated)

• Advantages

  • High throughput (96/384-well plates); amenable to automation and robotic liquid handling.

  • Highly sensitive (pg/mL range), depending on antibodies and detection chemistry.

  • Fully quantitative with proper standards.

  • Safe; no radioactivity.

  • Relatively low sample volume (typically 50\,\mu L per well).

• Disadvantages

  • Requires high-quality, specific antibody pairs; cross-reactivity → false results.

  • Provides no molecular weight information (cannot distinguish isoforms).

  • Matrix interference (heterophilic antibodies, complement) can cause artifacts.

  • Each assay detects only one analyte (unless multiplexed platform used).

When to Use ELISA

• Sample: fluids (serum, plasma, CSF, urine), culture supernatants, lysates where target concentration may be low (pg–ng/mL).

• Target location: secreted proteins, cytokines, hormones, antibodies in circulation; any soluble antigen.

• Ideal when absolute quantification of one analyte across many samples is needed.

Multiplex Immunoassay (Luminex® Technology)

• Goal: measure multiple analytes simultaneously (up to 100) in minimal sample volume.

• Bead principle

  • Polystyrene or paramagnetic beads internally dyed with distinct combos of red & infrared fluorophores.

  • Each bead region corresponds to a unique analyte (capture Ab coupled on surface).

• Assay steps

  1. Mix different bead regions in one well.

  2. Add sample → analytes bind their respective bead-attached capture Abs.

  3. Add biotinylated detection Abs (analyte-specific).

  4. Add streptavidin-phycoerythrin (PE) → binds biotin → fluorescent reporter.

  5. Read on dual-laser flow cytometer-based instrument.

  • Laser 1: classifies bead region (analyte ID).

  • Laser 2: quantifies PE intensity (analyte amount).

  1. Generate standard curve for each analyte within same run.

Multiplex Advantages / Disadvantages

• Advantages

  • Simultaneous quantification of many proteins in \le 50\,\mu L of sample.

  • Cost-effective per analyte vs multiple single ELISAs.

  • Comparable sensitivity, accuracy, reproducibility to ELISA when validated.

  • Customisable panels (cytokines, signaling proteins, etc.).

• Disadvantages

  • Requires specialised, more expensive instrumentation (Luminex reader).

  • Necessitates rigorous validation of each antibody pair in multiplex context (cross-reactivity, interference).

  • Dynamic range must overlap among analytes or require sample dilution adjustments.

Technique Selection Decision Points

• Need molecular weight / isoform info? → Western Blot.

• High throughput, single analyte quantification? → ELISA.

• Multiple analytes, low sample volume, medium-high throughput? → Luminex / other multiplex.

• Quantification accuracy priority → ELISA or Luminex with proper standards.

• Verification of protein identity after gene editing or cleavage events → Western Blot.

Ethical, Safety, & Practical Notes

• Radioimmunoassay (historical) involves radioactivity → regulatory burdens; ELISA & multiplex avoid this.

• Chemiluminescent substrates generate transient signals; imaging must occur promptly (ethical resource use: avoid repeat runs).

• Western blotting requires disposal of acrylamide (neurotoxin) and chemiluminescent reagents (hazardous waste).

• Multiplex assays reduce animal use for antibody production by maximising data per sample.

Numerical & Statistical References

• Sample volume for Luminex: \le 50\,\mu L.

• Temperature for native PAGE: 4^\circ C.

• Typical blocking reagents: 5\% skim milk; 1\% BSA.

• Housekeeping proteins commonly used: GAPDH (~36\,\text{kDa}), \beta-actin (~42\,\text{kDa}), tubulin (~55\,\text{kDa}).

Connected Learning Resources

• Alberts et al., “Molecular Biology of the Cell”, Ch. 8, §3 (pp. 487-490).

• Kuby Immunology 7th ed., Ch. 20 (pp. 659-662) – ELISA.

• Supplementary videos:

  • Protein isolation: https://www.youtube.com/watch?v=GJJGNOdhP8w

  • PAGE run: https://www.youtube.com/watch?v=JcN0EkcHrKk

  • Transfer & visualisation: https://www.youtube.com/watch?v=IoVzpL_heFo

  • Gel electrophoresis animation: https://www.youtube.com/watch?v=IWZNGpC8U

  • ELISA demo: https://www.youtube.com/watch?v=lUWpWKVcmc4

  • Luminex workflow: https://www.youtube.com/watch?v=v4kH4d3dLwg