How are antibodies used in a lab? Western blot, IHC, IF, ELISA and more

An antibody is a protein that binds a specific region, called an epitope, on its target. In research that specificity is used to find one protein among thousands. The antibody either carries a label itself, such as an enzyme or a fluorophore, or is detected by a labelled secondary antibody that recognises it.

Primary antibodies bind the target. They are made in a host species such as rabbit or mouse and are either monoclonal, recognising one epitope, or polyclonal, recognising several. The monoclonal vs polyclonal guide covers that choice.

Proteins from a lysate are denatured, separated by size with SDS-PAGE and transferred to a membrane. The membrane is blocked with milk or BSA, incubated with the primary antibody, then an HRP-labelled secondary, and developed with a chemiluminescent substrate.

A good result is a single band at the expected molecular weight. Because the protein is denatured, western blot works best with antibodies against linear epitopes. A lane with a prestained protein ladder lets you check transfer and size the band, and a loading control antibody, such as one against β-actin, GAPDH or α-tubulin, shows that each lane received the same amount of protein.

The choice of extraction buffer comes first. The lysis buffer guide compares RIPA, NP-40 and native buffers.

IHC shows where a protein sits inside tissue. Formalin-fixed, paraffin-embedded sections are dewaxed and rehydrated, and the cross-links formed by formalin are partly reversed by heating in a retrieval buffer. The primary antibody is applied, then an HRP-based detection system that deposits a brown DAB precipitate, and the section is counterstained with haematoxylin.

Antigen retrieval makes or breaks IHC. The Tris-EDTA pH 9 retrieval protocol explains when to use it instead of citrate pH 6.

Immunofluorescence uses fluorophore-labelled antibodies, so several targets can be imaged at once in different colours. For cultured cells, sometimes called immunocytochemistry, cells are typically fixed with 4% paraformaldehyde, permeabilised so antibodies can reach inside, blocked, stained and mounted with a nuclear counterstain such as DAPI.

ELISA turns antibody binding into a number. In a sandwich ELISA, a capture antibody on a plate holds the target and a second antibody detects it, giving a concentration against a standard curve. The guide to ELISA types explains the formats, and ready-made ELISA kits supply validated antibody pairs for many targets.

Flow cytometry measures fluorescent antibody binding on thousands of single cells per second, counting cell populations by their surface or intracellular markers. Antibodies are usually directly conjugated to fluorophores, and Fc receptor blocking reduces non-specific binding.

Immunoprecipitation (IP) uses an antibody bound to protein A or G beads to pull a target out of a lysate. Co-immunoprecipitation pulls down the target’s binding partners too, which needs a gentle buffer that keeps complexes intact.

• Positive control: a cell line or tissue known to express the target.
• Negative control: a sample known not to express it, ideally a knockout or knockdown.
• Secondary-only control: omit the primary to reveal background from the secondary.
• Isotype control: an antibody of the same class with no relevant target, for flow cytometry and IHC.
• Loading control: a housekeeping protein to normalise western blots.

Dilution matters as much as the controls. Titrate each new antibody, and dilute it in an antibody diluent, a buffered protein solution such as PBS or TBS with BSA, rather than plain buffer. The protein keeps a dilute antibody stable and lowers background.

Control antibodies, loading controls and secondaries are part of the antibody range, alongside the primaries.