A heterobifunctional (N-hydroxy succinimide) ester diazrine crosslinker, (NHS-diazirine, succinimidyl 4,4′-azipentanoate, Pierce Biotechnology) was conjugated to the primary amines contained in ELP chains as previously reported.[33 (link)] Briefly, the NHS-diazirine was dissolved in dimethyl sulfoxide (0.5–1 g/mL) and mixed with a solution of ELP (50 mg/mL in phosphate buffered saline (PBS)) to a final stoichiometric ratio of 1:1 functional groups. After reaction, the solution was dialyzed, lyophilized, and stored at 4 °C. All subsequent references to ELP refer to this diazirine-modified photocrosslinkable form.
Nhs diazirine
Manufactured by Thermo Fisher Scientific
NHS-diazirine is a heterobifunctional crosslinking reagent used for covalent protein-protein and protein-ligand conjugation. It contains an N-hydroxysuccinimide (NHS) ester group for primary amine labeling and a photo-activatable diazirine group for non-specific photo-crosslinking.
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Lab products found in correlation
4 protocols using nhs diazirine
1
Photocrosslinkable Diazirine-Modified ELP
2
UV-Induced Protein-Protein Crosslinking
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Samples (100 μg) of GADD45β and MKK7_KD were dissolved in 10 mM Sodium Phosphate buffer pH 8.0 to a final concentration of 10 μM. The amine-reactive cross-linking reagent NHS-Diazirine (SDA) was used as photoactivable reagent (Pierce Biotechnology, ThermoFisher). Preliminary experiments were carried out to determine the optimal protein concentrations and cross-linking reagent-to-protein molar ratios. Following the optimization of the reactions, SDA was dissolved in 10 mM DMSO pH 8.0 and added in a first step to the MKK7_KD protein solution (270 μL) with a 20-fold excess (mol/mol) of the cross-linker. Following incubation for 1 h at room temperature, reactions were quenched by adding 1 M Tris-HCl buffer pH 8.0 at a final concentration of 50 mM. Then, the unreacted cross-linker was removed by microfiltration by using Corning Spin-X UF Concentrators MWCO 5000 (Millipore). Protein quantification was performed by Bradford assay. For the second step, equal moles (700 pmol) of GADD45β were added to the SDA-labeled MKK7_KD to perform the UV-cross-linking reaction (40 μL final volume). Protein mixtures in clear, V-bottom 96-well plates were irradiated at 365 nm for 15 min on ice by using a Black-Ray UVP B-100 A, 100 W lamp (UVP).
3
Photoreactive Peptide Diaz-ES-VWFIII Synthesis
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Resin beads bearing end-stapled VWFIIINle (8.3 × 10−6 mol) were conditioned in 10 ml of dry DMF away from light for 5 min. DIEA (5.7 μl, 3.3 × 10−5 mol) and NHS-Diazirine (5.63 mg, 2.5 × 10−5 mol, Life Technologies) were added to the mixture. The reaction was left overnight at room temperature in the dark and the resin was washed with DCM twice, MeOH twice and DCM, to give the photoreactive peptide Diaz-ES-VWFIIINle.
4
Preparation of End-Stapled Triple-Helical Peptide
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The end-stapled triple-helical peptide (6.5 mg, 6.15 × 10−7 mol) was dissolved in 100 μl of dry DMF away from light. DIEA (0.2 μl, 1.23 × 10−6 mol) and NHS-Diazirine (0.3 mg, 1.23 × 10−6 mol, Life Technology) were added to the mixture. The reaction was left overnight at room temperature away from light. 1 ml of cold diethyl ether was added, resulting in the precipitation of a white solid. The mixture was filtered, the precipitate washed with 1 ml of cold diethyl ether, dissolved in H2O/ACN 95/5 and 0.1% TFA and freeze dried to yield 6.1 mg of white powder. The resulting product was further purified by dialysis in 0.01 M acetic acid (AcOH) during 4 h three times using a Float-A-Lyzer 500-1000 MWCO (Spectrum-Labs). After freeze-drying, 5.7 mg (5.4 × 10−7 mol) of the expected product was recovered as a white powder (87% yield).
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