If you’ve ever mixed up Northern, Southern, and Western blots, you’re not alone. The names follow a pun on Edwin Southern’s original technique. Each detects a different type of biomolecule, but they share a common workflow: separate by size, transfer to membrane, probe specifically.
Quick reference
| Blot | Detects | Probe | Separation |
|---|---|---|---|
| Southern | DNA | Labeled DNA/RNA | Agarose gel |
| Northern | RNA | Labeled DNA/RNA | Denaturing agarose gel |
| Western | Protein | Antibody | SDS-PAGE |
Southern blot
Developed by Edwin Southern in 1975. DNA is digested with restriction enzymes, separated by agarose electrophoresis, transferred to nitrocellulose or nylon, and detected with a labeled DNA probe complementary to the target sequence.
Classic uses: detecting gene rearrangements, RFLP analysis, transgene copy number, confirmation of gene targeting in knockout mice. Largely replaced by PCR, qPCR, and NGS for routine work, but still used in some clinical contexts (e.g., antibody gene rearrangement in lymphoma diagnosis).
Northern blot
Named in pun on Southern’s name. RNA is separated on a denaturing agarose gel, transferred to a membrane, and probed with a labeled RNA or DNA complementary to the transcript.
Northerns directly visualize transcript size and isoforms — useful for identifying alternative splice forms, premature termination, and degradation. Largely replaced by RT-qPCR (quantification) and RNA-seq (size + sequence). Still occasionally used to confirm transcript identity, especially when isoform size matters.
Western blot
Detects proteins. Proteins are denatured and separated by SDS-PAGE, transferred to PVDF or nitrocellulose, and detected with primary antibody followed by enzyme-conjugated secondary antibody. The membrane is developed with a chromogenic or chemiluminescent substrate.
Westerns remain a daily workhorse — there’s no equivalent technique that combines size separation with protein-specific detection at comparable cost.
Modern alternatives
- For DNA: qPCR for copy number, NGS for sequence-level detail, FISH for cytogenetics
- For RNA: RT-qPCR for quantification, RNA-seq for genome-wide expression, in situ hybridization (RNAscope) for spatial localization
- For protein: Mass spectrometry for unbiased proteomics, ELISA for absolute quantification, Simple Western (capillary-based) for automated workflows
Why blots persist despite alternatives
- Direct visualization. A blot shows you size and abundance simultaneously — no software interpretation required.
- Validation step. A western confirms a protein-level change that an RNA-seq result implies.
- Single-target focus. When you care about one specific molecule, blots are cheap and direct.
Mnemonic
The pun goes South to North to West: Southern for DNA (the original), Northern for RNA (next), Western for protein (the latest of the three classics).
Each technique has been augmented by faster, more sensitive methods, but Westerns in particular remain irreplaceable for many labs. Knowing which blot is which lets you interpret older literature critically.
