Photo-reactive crosslinkers are widely used for nonspecific bioconjugation. While numerous options exist, the two most common photo-reactive chemical groups are diazirines and aryl-azides.  

Photoreactive reagents like aryl azides and diazirines are most often used as heterobifunctional crosslinkers to capture binding partner interactions. They are typically used in the following manner:

  1. A purified bait protein is labeled with the crosslinker using the amine- or sulfhydryl-reactive end
  2. The labeled protein is then added to a lysate sample and allowed to bind its interactor
  3. Finally, photo-activation with UV light initiates conjugation via the phenyl azide group

Aryl azides have been the most popular photoreactive chemical group used in crosslinking and labeling reactions. When exposed to UV light (250-350nm), aryl azide forms a nitrene group that can do any of the following (1) initiate an addition reaction with the double bonds, (2) insertion into a C–H or N–H sites, (3) undergo a ring expansion to react with a nucleophile (e.g., primary amines).

Diazirines are a newer class of photoactive chemical group suitable for crosslinking and labeling reagents. The diazirine (azipentanoate) group has better photostability than phenyl azide groups, and is easily and efficiently activated with long-wave UV light (330-370nm).

Choose the right photoreactive crosslinker for your experiment

Reactive Groups Products Spacer Arm (Å) Cleavable by? Water-soluble ? Membrane permeable?
NHS ester/ aryl azide ANB-NOS 7.7Short No No No
Sulfo-SANPAH 18.2 Long No Yes No
NHS ester/ diazirine SDA 3.9 Short No No Yes
Sulfo-SDA 3.9 Short No Yes No
LC-SDA   12.5 Mid No No Yes
Sulfo-LC-SDA 12.5 Mid No Yes No
SDAD   13.5 Mid Thiols No Yes
Sulfo-SDAD 13.5 Mid Thiols Yes No

Featured photoreactive crosslinkers and accessories

This heterobifunctional, photoreactive crossslinker covalently links two target groups:  the succinimidyl ester chemically reacts with primary amines and upon UV illumination, the photoreactive component reacts with nucleophiles or forms C-H insertion products.

Diazirine analogs of leucine and methionine for the expression of proteins in cell culture that will crosslink their protein interactors upon UV-light activation in vivo.

This heterobifunctional, photoreactive crossslinker covalentlys link two target groups: following a mild reduction, the dithiol can react form a disulfide with other thiols and upon UV illumination, the photoreactive component reacts with nucleophiles or forms C-H insertion products.

Chemical-tag purified proteins at primary amines that covalently crosslink and transfer biotinylation to protein interaction binding partners.

An amine-to-DNA crosslinker, composed of NHS-ester and psoralen reactive groups, which conjugates to DNA via light-activated intercalation next to pyrimidine bases.

Compact, UV-lamps with 252 nm, 302 nm and 365 nm short to long wavelength bulbs for use with photoactive crosslinkers and UV-crosslinking methods.

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