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Short answer: A cross linker (crosslinker) is a molecule that forms covalent bonds between two or more other molecules or sites. In a research lab the term usually means a chemical reagent that joins proteins or other biomolecules, or a UV instrument that fixes nucleic acids to a membrane. The reagent you need depends on which groups you target, how far apart they are, and whether the link must be reversible.
This page is general reference for lab and research staff, not protocol advice or medical advice.
Definition and scope
In the protein-chemistry literature, a crosslink is a physical or chemical bond that associates reactive groups in a polymer network (Jayachandran et al., Molecules 2022). The same word covers three things people search for, so it helps to separate them first:
- Chemical crosslinker reagent: a molecule with reactive groups that bridges functional groups on proteins or other biomolecules. This is the main subject of this page.
- UV crosslinker instrument: a UV irradiation cabinet used to link DNA or RNA to a blotting membrane (see the FAQ below).
- Polymer and coating crosslinkers: agents that cure inks, paints, leather finishes and similar materials. These are industrial chemistry and are outside this page.
Spelling: crosslink, cross link and cross-link are all in use for the noun and the verb, and a cross linker is the agent that makes the link. This page uses “crosslinker” and treats the other forms as the same thing.
How crosslinkers are used in practice
Crosslinkers are used to label, immobilise or stabilise proteins and to capture which molecules sit close together. According to Thermo Fisher’s protein crosslinking support documentation, a crosslinker is built from reactive groups at its ends and, usually, a spacer arm between them. Two design choices matter most.
Homobifunctional versus heterobifunctional
Homobifunctional crosslinkers have identical reactive groups at either end of the spacer arm. They work in one step and give a snapshot of what was close together, with less precision. Heterobifunctional crosslinkers have different reactive groups, which allows a one-step or a controlled two-step reaction and reduces polymerisation and self-conjugation.
Spacer arm
Spacer arms range from zero length (the groups are joined directly) to more than 100 angstroms. Thermo Fisher notes that longer arms have greater flexibility and reduced steric hindrance; linear hydrocarbon arms are generally hydrophobic and can cross membranes, whereas sulfonate or PEG groups improve water solubility and can make a reagent cell-impermeant, so it labels only the cell surface. The carbodiimide EDC is the standard zero-length example: it joins a carboxyl group directly to an amine and leaves no spacer, though it can cause random polymerisation.
Reactive groups and what they target
This table is built only from the Thermo Fisher support page and the Jayachandran review; conditions are the ranges Thermo Fisher states.
| Reactive group | Targets | Condition stated | Bond or note |
|---|---|---|---|
| NHS ester | Primary amines | pH 7.2-8.5 | Amide bond |
| Imidoester | Primary amines | About pH 10 | Amidine; keeps the positive charge |
| Maleimide | Sulfhydryls | Near-neutral pH | Thioether |
| Haloacetyl | Sulfhydryls | pH 7.2-9 | Thioether |
| Pyridyldisulfide | Sulfhydryls | – | Disulfide; cleavable with reducing agents |
| Carbodiimide (EDC) | Carboxyl to amine | – | Zero-length; water soluble; often random polymerisation |
| Hydrazide | Carbonyls | pH 5-7 | Hydrazone; carbonyls on glycoproteins can be made with sodium meta-periodate |
| Aryl azide, diazirine | Nonselective (photoreactive) | UV light; diazirine 330-370 nm | Used when the target residue is unknown; Thermo Fisher states diazirines have better photostability than phenyl azides |
Which amino acids can cross-link?
With chemical crosslinkers the targets are the side-chain and terminal groups the reagent chemistry can reach: primary amines (the N-terminus and lysine), carboxyls (the C-terminus, aspartate and glutamate), the thiol of cysteine, and carbonyls on the carbohydrates of glycoproteins (Jayachandran et al., 2022). Photoreactive groups are the exception because they react nonselectively when activated.
Choosing a crosslinker: a selection checklist
Thermo Fisher lists eight selection factors; this checklist groups them into practical questions. Each answer changes which reagent is suitable.
| Question | What it changes |
|---|---|
| Are you working on live cells? | Live-cell work needs a cell-permeable crosslinker; charged sulfo-NHS reagents label the surface only. |
| Is the target residue known? | If it is unknown, use a photoreactive crosslinker. |
| Should the reaction start spontaneously or on a trigger? | Spontaneous groups react on mixing; photoreactive groups react when exposed to light. |
| Must the linked molecules be separated again? | A cleavable crosslinker is recommended if you need to dissociate the linked molecules. |
| What solvent will you use? | Hydrophobic reagents are dissolved in DMSO or DMF; sulfonated and PEG reagents go into aqueous buffer. |
| How many reactive sites does the protein have, and what is the readout? | Use a lower crosslinker-to-protein ratio for proteins with many targets and a higher one for few. Also consider the downstream gel, western blot or mass spectrometry readout. |
Where it matters for labs
Crosslinking sits behind several techniques that already have pages on this site: co-immunoprecipitation workflows, cell fixation, expansion microscopy and hydrogels, whose networks are held together by crosslinks. Labelling chemistry is also related to biotinylation. Safety and handling vary by reagent: the Jayachandran review describes glutaraldehyde as toxic, and genipin, a natural crosslinker that reacts with primary amines, as less toxic. Always follow the manufacturer’s safety data sheet.
Common misunderstandings
- “Crosslinker” always means a reagent. A UV crosslinker is an instrument, and polymer crosslinkers are a separate industrial category.
- Every crosslinker is reversible. Only cleavable designs, such as those with a disulfide spacer, can be separated again.
- Longer is always better. Spacer length is a trade-off between flexibility, solubility and what the reagent can reach.
- Crosslinking is selective by default. Amine reagents react with any accessible primary amine; EDC can polymerise proteins randomly.
FAQ
Is it crosslink or cross link?
Both appear in use, along with “cross-link”. They mean the same thing; this page standardises on “crosslinker”. What is a crosslink, what is a cross link and what is a crosslinker all refer to the bond (or the agent making it) joining molecules, as described above.
What is a cross linker?
A molecule, usually with a reactive group at each end of a spacer arm, that covalently joins two molecules or sites. See the definition and selection sections above.
What is a UV crosslinker used for?
In molecular biology, a UV crosslinker is an instrument that irradiates a blotting membrane to link DNA or RNA to it. The manufacturer Analytik Jena describes its UVP CL-3000 (254 nm) as a UV irradiation system for nucleic acid linking to membranes in Southern, Northern, dot and slot blot applications. See Southern blot and Northern blot. It is a different object from the chemical reagents above.
What is cross linkage in DNA?
The term is used in several senses, including linking nucleic acids to a membrane with UV light (above). Other kinds of DNA crosslinking are a separate topic that this page does not cover; the reagent chemistry here concerns proteins.
Which amino acids can cross-link?
See the section above: lysine and the N-terminus (amines), aspartate, glutamate and the C-terminus (carboxyls), cysteine (thiol), and glycoprotein carbonyls; photoreactive reagents are nonselective.
Sources
- Thermo Fisher Scientific, Protein Labeling, Crosslinking and Modification Support: Getting Started, thermofisher.com (read 2026-11-07).
- Jayachandran B. et al., Insights on Chemical Crosslinking Strategies for Proteins, Molecules 2022, 27(23):8124, PMC9738610 (read 2026-11-07).
- Analytik Jena, UVP Crosslinker CL-3000, analytik-jena.com (read 2026-11-07).
