Blotting Techniques

4 min read Updated Apr 18, 2026

A gel tells you how big your fragments are but not which one is the specific molecule you care about. Blotting adds specificity: transfer the gel to a membrane, then probe the membrane with a labeled molecule that binds only your target.

Three main blots, each for a different target molecule type.

The SNoW DRoP Mnemonic

Southern Blot (DNA)

Workflow:

Cut genomic DNA with a restriction enzyme.
Run on a gel to separate fragments by size.
Transfer the DNA onto a membrane (nitrocellulose or nylon).
Incubate the membrane with a labeled DNA probe (radioactive or fluorescent) complementary to the target sequence.
Wash off unbound probe, then detect where the probe stuck.

A single band on the resulting film tells you the target sequence is present in the genome and at what size the restriction enzyme cut it. Southern blotting was historically used to detect gene rearrangements, copy number variations, and specific mutations. It has largely been replaced by PCR and sequencing.

Northern Blot (RNA)

Essentially the same procedure, but starting with RNA. Used to detect and size a specific mRNA. Tells you whether a gene is expressed in a tissue, whether alternative splicing produces multiple transcript sizes, and whether the transcript is degraded. Like Southern, it has been largely supplanted by quantitative methods (qPCR, RNA-seq).

Western Blot (Protein)

Workflow:

Run proteins on SDS-PAGE.
Transfer to a membrane.
Probe with a specific antibody against the target protein.
A secondary antibody (attached to an enzyme or fluorophore) detects the primary antibody.
A chemiluminescent substrate reveals where the primary antibody bound.

Western blotting remains the gold standard for confirming that a specific protein is present (and at what size) in a sample. It is used throughout molecular and clinical biology.

Hybridization Probes vs. Antibodies

The key difference between Southern/Northern and Western blots:

Southern and Northern use complementary nucleic acid probes (DNA or RNA) that base-pair with the target. Specificity comes from sequence complementarity.
Western uses antibodies that recognize a specific protein epitope. Specificity comes from the protein-protein interaction.

What does each of Southern, Northern, and Western blot detect?

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Southern detects DNA. Northern detects RNA. Western detects Protein. The mnemonic SNoW DRoP captures this: Southern-DNA, Northern-RNA, Western-Protein.

What is the fundamental probe difference between a Southern blot and a Western blot?

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Southern blots use a complementary nucleic acid (DNA or RNA) probe that base-pairs with target DNA. Western blots use antibodies that recognize specific protein epitopes. The recognition principle differs: hybridization vs. protein-protein binding.

Why is Western blot often used as the confirmatory test after a positive ELISA screen for HIV?

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ELISA is highly sensitive but can yield false positives. Western blot separates viral proteins by size, then uses the patient's antibodies to confirm that bands at specific sizes (gp120, gp41, p24) are present. Multiple specific bands give high specificity. Screen with the sensitive test (ELISA), confirm with the specific test (Western blot).