Azidobutyric acid NHS ester

This plan only provides a guide, please modify it to meet your specific needs.
1. Solution preparation
 
(1) Working solution: Weigh the solid and dilute it with DMSO to the required working concentration.
 
Note: Store solids at -20°C or -80°C protected from light. Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration yourself to explore. The working fluid must be prepared and used immediately.
 
 
2. Use Azidobutyric acid NHS ester to activate the elastin-like peptide and conjugate it to an alkyne-modified dye through click chemistry to achieve labeling ^[1] (from the literature, for reference only)
 
(1) Preparation of Azidobutyric acid NHS ester solution: Dilute Azidobutyric acid NHS ester in DMSO to a final concentration of 20 mM.
(2) Activation of elastin-like peptide (ELP): Dissolve ELP in PBS, adjust the pH value to 6.8–7.0, mix ELP with a 2-fold excess of Azidobutyric acid NHS ester, and incubate at room temperature for 12 hours.
(3) Remove residual Azidobutyric acid NHS ester: Put the sample into a 10 kDa dialysis cassette and dialyze at 4°C for 12 hours.
(4) Conjugation by click chemistry: Mix alkyne-modified dye and activated ELP at a molar ratio of 1:1 (dye:ELP), add 1 mM TBTA (GC45003), 10 mM TCEP (GF02960), 10 mM CuSO4 to the mixture (final concentration) and incubate at 4°C for 12 hours.
(5) Remove the remaining connecting chains: Put the sample into a 10 kDa dialysis cassette and dialyze at 4°C for 12 hours.
 
Note: This experiment provides a reference for using Azidobutyric acid NHS ester to activate elastin-like peptides and conjugate them with alkyne-modified dyes through click chemistry to achieve labeling. The reaction concentration and reaction time can be optimized according to actual conditions. Operations should be performed in a sterile environment to prevent contaminants from interfering with the reaction. Avoid direct contact with reaction reagents.
 
References:
[1] Vogele K, Frank T, Gasser L, et al. Towards synthetic cells using peptide-based reaction compartments[J]. Nature Communications, 2018, 9(1): 3862.