Western Blotting (Protein Immunoblot)

Definition

• Medical Microbiology 9e

Principle

1. Separation of proteins by molecular weight (size)
2. Transfer of separated proteins to a solid membrane support
3. Detection of the target protein using antibody probes

Step-by-Step Procedure

1. Sample Preparation

• Cells or organisms are mechanically or chemically disrupted to release proteins
• Proteins are denatured with a strong reducing agent (e.g., SDS - sodium dodecyl sulfate)
• SDS coats proteins with a uniform negative charge, so separation is purely by size

2. Gel Electrophoresis (SDS-PAGE)

• The protein mixture is loaded into a polyacrylamide gel
• An electric current drives proteins through the gel - smaller proteins migrate faster and travel further
• This separates proteins into distinct bands by molecular weight

3. Transfer (Blotting)

• The separated protein bands are transferred electrophoretically from the gel onto a nitrocellulose or nylon membrane
• Transfer occurs by passing an electric current through a "sandwich" (gel + membrane) submerged in transfer buffer - proteins migrate toward the positive electrode onto the membrane
• The membrane is then blocked (usually with milk proteins) to prevent nonspecific antibody binding

4. Primary Antibody Incubation

• The membrane is incubated with the patient's serum or a specific primary antibody
• The primary antibody binds to its target protein on the membrane
• Polyclonal antibodies recognize multiple epitopes; monoclonal antibodies recognize a single epitope

5. Secondary Antibody Detection

• A labeled secondary antibody (anti-human IgG) is added - it binds to the primary antibody
• The secondary antibody carries a detection label: enzyme, fluorescent probe, or radioactive isotope
• Enzyme-labeled antibodies convert a substrate to produce a colorimetric or chemiluminescent signal
• The pattern of bands reveals which proteins the antibodies bind to
• Medical Microbiology 9e; Dermatology 2-Volume Set 5e; Jawetz Melnick & Adelbergs Medical Microbiology 28E

The Antibody System

• Enzyme (e.g., HRP) - produces color or chemiluminescence on substrate
• Fluorescent probe - produces light signal
• Radioactive isotope (e.g., I-125, older method)

Purpose / Uses

Clinical Applications

1. HIV - historically used as confirmatory test after positive ELISA/EIA. A positive Western blot in the right context confirms HIV infection. The CDC no longer recommends Western blot for routine HIV diagnosis (replaced by newer supplemental assays), but it remains conceptually relevant.
2. Lyme disease - a two-tier approach: ELISA first, then Western blot to confirm a positive/equivocal ELISA result. The IgM Western blot has a high false-positive rate when pretest probability is low. - Textbook of Family Medicine 9e
3. Prion diseases - used in Creutzfeldt-Jakob disease (CJD) diagnosis
4. HIV-2 - indeterminate HIV-1 Western blot results may indicate HIV-2 infection, requiring a separate HIV-2 Western blot - Jawetz 28E

Benefits

• Simple and sensitive method to detect and quantify proteins
• Can determine the molecular weight of a specific protein relative to standard molecular weight markers
• Can differentiate specific reactivity from nonspecific (negative) reactions

Limitations / Sources of Error

• Requires a specific antibody that recognizes the target protein (unavailable for some antigens)
• May produce high background of nonspecific staining
• Proteins may degrade during extraction
• IgM Western blot results can give false positives when pretest probability is low (e.g., Lyme disease)
• Indeterminate results occur (e.g., HIV-2 cross-reactivity with HIV-1 test)
• Dermatology 2-Volume Set 5e

Related / Alternative Techniques

• Dermatology 2-Volume Set 5e

Blot Family Summary

Southern Blotting

Definition

• Emery's Elements of Medical Genetics and Genomics; Schwartz's Principles of Surgery 11e

Core Principle

1. At the level of restriction enzyme recognition (where cuts are made)
2. By the size of the resulting DNA fragment
3. By hybridization of a DNA probe to a specific locus (sequence identity)

• Jawetz Melnick & Adelbergs Medical Microbiology 28E

Step-by-Step Procedure

Step 1: DNA Extraction

• DNA is extracted from the sample (e.g., patient's white blood cells, bacterial colony)
• The entire genomic digest may yield ≥10⁶ fragments

Step 2: Restriction Enzyme Digestion

• DNA is cleaved with a restriction endonuclease - a highly specific bacterial enzyme that recognizes a unique palindromic sequence (usually 4-8 bp, e.g., GAATTC)
• The pattern of fragments produced is unique to each gene sequence
• Wild-type and mutant DNA are discriminated by the size of resulting fragments

Step 3: Gel Electrophoresis

• The DNA fragments (all negatively charged) are loaded onto an agarose gel
• An electric current drives them through the gel - smaller fragments migrate faster and travel farther
• A DNA ladder (standard fragments of known size) runs alongside to allow sizing of unknown bands
• The gel is stained with ethidium bromide and photographed with a ruler alongside

Step 4: Denaturation

• The double-stranded DNA in the gel is denatured with alkali (e.g., NaOH), converting the fragments to single-stranded DNA
• This is necessary for probe hybridization in the next steps

Step 5: Transfer (Blotting) to Membrane

• The single-stranded DNA fragments are transferred from the gel to a nitrocellulose or nylon membrane, preserving their relative positions exactly
• Transfer occurs either by:
  • Capillary diffusion - buffer wicks upward through the gel, carrying DNA fragments onto the membrane above (via a "sandwich" of filter paper, gel, membrane, and paper towels)
  • Electrophoretic transfer - electric current drives DNA onto the membrane
• The membrane permanently binds the single-stranded DNA

Step 6: Probe Hybridization

• A single-stranded DNA probe - with a known sequence complementary to the target DNA - is added to the membrane
• The probe is labeled with:
  • ³²P radioactive isotope (traditional method) - detected by autoradiography/X-ray film
  • Digoxigenin (non-radioactive) - detected by chemiluminescence (safer, faster)
• The probe hybridizes only to fragments with a complementary sequence

Step 7: Detection

• Radioactive method: Membrane is exposed to X-ray film (autoradiography) - the probe-bound bands appear as dark bands on the film
• Chemiluminescent method: Generates a light signal detected on film or digitally
• Lippincott Biochemistry 8e; Emery's Elements of Medical Genetics and Genomics; Creasy & Resnik's Maternal-Fetal Medicine; Schwartz's Principles of Surgery 11e

What Southern Blotting Can Detect

• Lippincott Biochemistry 8e

Clinical Applications

• Creasy & Resnik's Maternal-Fetal Medicine; Jawetz 28E

Restriction Fragment Length Polymorphism (RFLP)

• Linkage analysis and gene mapping
• Prenatal diagnosis of hereditary conditions
• Forensic DNA profiling

Limitations

• Requires large amounts of DNA (relatively)
• Labor-intensive and difficult to automate → expensive
• Low throughput compared to PCR-based methods
• For point mutations not at restriction sites, Southern blotting alone cannot detect them
• Has been largely replaced by PCR for most applications, though it remains the gold standard for detecting large repeat expansions (e.g., Fragile X)
• Creasy & Resnik's Maternal-Fetal Medicine

Blot Family Comparison

Note from Lippincott Biochemistry: "Northern" and "Western" are facetious extensions of the name - they do not refer to any person's name, nor to points of the compass.