Western blot & other blotting techniques

Western detects protein with antibodies; Southern detects DNA and Northern detects RNA with complementary probes.

Western blot: detect a particular protein

Separate proteins, usually by SDS-PAGE, then transfer them to a nitrocellulose or PVDF membrane. Transfer preserves the pattern of separated bands.
Block unoccupied membrane sites with a protein solution, such as BSA or milk proteins, to reduce nonspecific binding.
Apply a primary antibody that recognizes the target. Wash, then apply a labeled secondary antibody that recognizes the primary antibody; wash again.
Detect the label by light emission, fluorescence, or color. A signal’s position estimates apparent size; its intensity can reflect abundance when loading, exposure, and signal range are controlled.

Proteins are separated, transferred to a membrane, detected with antibodies, and visualized as bands. — **Western blot workflow** Khan Academy · CC BY-NC-SA 4.0 · Original image ↗

Specificity matters. An unexpected band can reflect cross-reactivity, degradation, or a modified protein. Appropriate controls and a molecular mass ladder help interpret the result. Some methods directly label the primary antibody.

ELISA: detect or quantify an antigen

ELISA uses antibody binding on a solid surface, commonly a microplate, followed by an enzyme-generated signal. In a sandwich assay, a capture antibody binds the antigen and a detection antibody recognizes another site. Compare signal with standards to estimate concentration. ELISA does not provide the gel-based size information of a Western blot.

A plate-bound capture antibody binds antigen, followed by enzyme-linked detection and a substrate-generated signal. — **Antibody detection in an ELISA** Khan Academy · CC BY-NC-SA 4.0 · Original image ↗

Southern and Northern blots

For a Southern blot, digest DNA into fragments, separate them by electrophoresis, denature the strands, and transfer them to a membrane. A labeled complementary probe hybridizes to the target sequence. Washing removes unbound probe before detection.

DNA is restriction-digested, separated, transferred, hybridized with a probe, washed, and detected. — **Southern blot workflow** BioLibreTexts, as credited by Khan Academy · Source attribution · Original image ↗

A Northern blot follows the same general separation–transfer–hybridization logic for RNA, usually without the restriction digestion used for DNA. It can reveal transcript size and relative abundance. Probes may use radioactive labels such as P-32 or nonradioactive labels.

Remember the targets. Southern → DNA; Northern → RNA; Western → protein. Nucleic acid blots use complementary base pairing; Western blots use antigen–antibody recognition.

20 terms, alphabetically ordered

Antibodyantibodies: An immune protein that specifically binds a molecular target called an antigen.
Blocking: Covering unoccupied binding sites on a membrane or plate to reduce nonspecific interactions.
Bovine serum albuminBSA: A protein commonly used to block unoccupied membrane binding sites and reduce nonspecific antibody binding.
Deoxyribonucleic acidDNA: The nucleic acid that stores genetic information used to specify RNA and protein sequences.
Electrophoresis: Movement of charged molecules in an electric field, used to separate molecules through a support such as a gel.
Enzyme-linked immunosorbent assayELISA: An antibody-based assay performed on a solid surface, commonly a microplate, to detect or quantify an antigen. It does not provide gel-based size information.
Hybridization: Pairing of complementary nucleic acid strands, used for sequence-specific detection.
Molecular mass ladderladder · molecular weight marker: A mixture of proteins of known masses, run beside samples to estimate their apparent sizes.
Northern blotNorthern blotting: Detection of a specific RNA sequence after gel separation and transfer, using a complementary labeled probe.
Phosphorus-32P-32 · ³²P: A radioactive isotope used to label nucleic acid probes.
Polyacrylamide gel electrophoresisPAGE · native PAGE: Electrophoretic separation using a polyacrylamide gel. Native PAGE preserves much of a protein’s native structure.
Polyvinylidene fluoridePVDF: A polymer used to make membranes that bind proteins during Western blotting.
Primary antibodyprimary antibodies: The antibody that binds directly to the target antigen.
Probeprobes: A labeled nucleic acid sequence that binds a complementary target through base pairing.
Ribonucleic acidRNA: A nucleic acid with roles in gene expression, regulation, and catalysis; messenger RNA carries protein-coding information.
Secondary antibodysecondary antibodies: An antibody that recognizes the primary antibody; often carries a detectable label or enzyme.
Sodium dodecyl sulfateSDS: An anionic detergent that unfolds proteins and coats them with negative charge. It does not itself reduce disulfide bonds.
Sodium dodecyl sulfate polyacrylamide gel electrophoresisSDS-PAGE: A denaturing protein separation method. SDS gives proteins a similar negative charge-to-mass ratio so migration depends mainly on molecular mass.
Southern blotSouthern blotting: Detection of a specific DNA sequence after gel separation and transfer, using a complementary labeled nucleic acid probe.
Western blotWestern blotting: Detection of a particular protein by gel separation, transfer to a membrane, and antibody binding.