Thermo Scientific Pierce NHS-Diazirine Crosslinkers combine proven NHS-ester and diazirine-based photoreaction chemistries to conjugate amine-containing molecules with nearly any other functional group via long-wave UV-light activation.
Succinimidyl-diazirine (SDA) reagents are a new class of crosslinkers that combine proven amine-reactive chemistry with an innovative and efficient diazirine-based photochemistry for conjugating amine-containing molecules to nearly any other functional group. The SDA crosslinkers include six compounds differing in spacer arm lengths, ability to cleave the crosslinked proteins, and presence or absence of a charged group for membrane permeability. Protein crosslinking is an important technique used to understand protein structure and to stabilize protein-protein interactions, and these SDA reagents extend the efficiency and range of interactions that can be explored by this approach.
- Heterobifunctional – succinimide ester group reacts with primary amines at pH 7 to 9 to form covalent amide bonds; diazirine (azipentanoate) group reacts efficiently with any amino acid side chain or peptide backbone upon activation with long-wave UV light (330-370nm)
- Controllable – two-step chemical crosslinking is activated using common laboratory UV lamps
- Easy to use – these crosslinkers are photo-stable under typical laboratory lighting conditions so there is no need to perform experiments in the dark
- Better than aryl azides – the diazirine photoreactive group has better photostability in normal light than phenyl azide groups of traditional photoreactive crosslinkers, yet the diazirine group is more efficiently activated by long-wave UV light
- Versatile – six different derivatives differing in spacer arm length, membrane permeability and cleavability for use in a variety of specific experimental strategies
Available Diazirine Crosslinkers:
- Short (3.9Å) and long (12.5Å) spacer arm varieties
- Membrane-permeable (NHS) and impermeable (Sulfo-NHS) varieties
- Non-cleavable and cleavable (disulfide spacer) varieties
for intracellular crosslinking)
for cell surface crosslinking)
Typical bifunctional amine- or sulfhydryl-reactive crosslinkers require specific amino acid groups (e.g., lysine or cysteine) at the correct distance on both proteins to capture protein interactions. The SDA crosslinkers circumvent this limitation by allowing specific labeling of one protein using the amine-reactive N-hydroxysuccinimide (NHS) ester followed by UV-activated crosslinking via the diazirine moiety to any amino acid side chain or peptide backbone of a second protein. Diazirine-based photocrosslinkers have better photostability than phenyl azide-based photocrosslinkers and are easily activated with long-wave UV light (330-370nm).
Mechanism of NHS-ester diazirine crosslinking (Figure, right). N-hydroxysuccinimide (NHS) esters react efficiently with primary amine groups (–NH2) in pH 7-9 buffer to form stable amide bonds upon release of NHS. Photoactivation of diarzirine with long-wave UV light (330-370nm) creates reactive carbene intermediates. Such intermediates can form covalent bonds throu addition reactions with any amino acid side-chain or peptide backbone at distances corresponding to the spacer arm lengths.
The NHS-ester diazirine derivatives (SDA, LC-SDA and SDAD) lack a charged group and are membrane-permeable. This property makes them ideal for intracellular and intramembrane conjugations. By contrast, Sulfo-SDA, Sulfo-LC-SDA and Sulfo-SDAD contain negatively charged sulfate groups that improve their water solubility and reduce cell membrane permeability, enabling their use for extracellular protein crosslinking. SDAD and Sulfo-SDAD also have a disulfide bond within the spacer that can be cleaved with reducing agents.
Capture and detection of intracellular protein homodimerization with Thermo Scientific NHS-Diazirine Crosslinkers (Figure, right). To demonstrate photo-reactive crosslinking of an intracellular protein complex with SDA reagents, our scientists examined protein interactions involving the early endosome antigen 1 (EEA1) protein in HeLa cells. EEA1 forms a homodimer through a coiled coil domain, binds to phospholipid vesicles and is involved in endosomal trafficking. Cells were incubated with various NHS-diazirine derivatives and treated with UV light. Cells were then lysed and analyzed by SDS-PAGE and Western blotting using an anti-EEA1 antibody.
After UV exposure, reduced mobility forms of EEA1 were observed only with samples treated with the SDA and SDAD and not a mock-treated control sample. Because EEA1 is an intracellular protein complex, it was not crosslinked by Sulfo-SDA, which does not permeate cell membranes. In addition, slower mobility, SDAD-crosslinked EEA1 could be cleaved within the spacer using the reducing agent dithiolthreitol (DTT). GAPDH detection was used to confirm equal protein loading.
Thermo Scientific NHS-Diazirine Reagents capture protein interactions more efficiently than other crosslinkers (Figure, right). We compared crosslinking of the heterodimeric sodium/ potassium (Na/K) ATPase after treating cells with a cleavable diazirine crosslinker (Sulfo-SDAD), a phenyl azide crosslinker (Sulfo-SANPAH) and the homobifunctional sulfo-NHS crosslinker (BS3).
Upon electrophoresis and Western blotting, significantly more crosslinked product of the cell-surface Na/Kb were observed with Sulfo-SDAD. Lack of heterodimer in the Sulfo-SDAD lane that was treated with dithiothreitol (DTT) indicates efficient to cleavage of the disulfide spacer arm in Sulfo-SDAD. GAPDH detection was used to confirm equal protein loading.
- Hermanson, G.T. (2008). Bioconjugate Techniques, 2nd Edition. Academic Press, New York. 1202 pages. (Part No. 20036).
- Gomes, A.F. and Gozzo, F.C. (2010). Chemical cross-linking with a diazirine photoactivatable cross-linker investigated by MALDI- and ESI-MS/MS. J. Mass. Spectrom. 45:892-9.
UV Lamps for crosslinking
Photo-reactive Amino Acids
Label Transfer Reagents