Azide Plus reagents is the most recent step in improving CuAAC reaction in complex media developed by scientists at Click Chemistry Tools. Azide Plus reagents contain a complete copper-chelating system in their structure, allowing for the formation of strong, active copper complexes that act simultaneously as both reactant and catalyst in the CuAAC reaction. This azide-copper complex reacts almost instantaneously with alkynes under diluted conditions. This unprecedented reactivity in the CuAAC reaction is of special value for the detection of low abundance targets, improving biocompatibility, and is also valuable for any other application where greatly improved S/N ratio is highly desired.

Learn more about azide plus reagents.

Click Reaction Protocol

This is a general protocol for labeling proteins in cell lysate through a copper-catalyzed click reaction using the Biotin Azide Plus reagent. We recommend using this protocol as a starting point for optimization of particular click chemistry procedures. We found that a 20 μM concentration of Biotin Azide Plus was sufficient to label all alkyne-tagged proteins in the cell lysate without causing a high background signal. The optimal final concentration of the Biotin Azide Plus reagent is sample dependent and may range from 5 μM to 50 μM. Final concentrations below or above this range are also possible, and should be optimized per the specific application.

1. Prepare the following click solutions:
– 100 mM THPTA ligand in water (100 mg of THPTA in 2.3 mL of water)
– 20 mM copper sulfate in water (dissolve 11.6 mg of copper II sulfate pentahydrate in 2.3 mL of water)
– 300 mM sodium ascorbate in water (dissolve 60 mg of sodium ascorbate in 1 mL of water)
– 1 mM Biotin Azide Plus reagent in DMSO (dissolve 1 mg of Biotin Azide Plus reagent in 425 μL of DMSO)

2. For each protein lysate sample, add the following to a 1.5 mL microfuge tube, then vortex briefly to mix.
– 50 μL of protein lysate (1-5 mg/mL) in protein extraction buffer
– 120 μL of Tris buffer
– 4 μL of Biotin Azide Plus stock solution (20 μM final concentration)

3. Add 10 μL of 100 mM THPTA solution, vortex briefly to mix.

4. Add 10 μL of 20 mM CuSO4 solution, vortex briefly to mix.

5. Add 10 μL of 300 mM sodium ascorbate solution to initiate click reaction, vortex briefly to mix.

6. Vortex continuously or rotate end-over-end for 30 minutes at room temperature.

7. Add the labeling reaction to 3 mL of cold (–20°C) methanol, 0.75 ml of Chloroform and 2.1 mL of water. Cool it to –20°C for 1 hour.
Note: cold (–20°C) acetone can be used in place of methanol:chloroform:water mixture.

8. Centrifuge for 10 minutes at 13,000-20,000 g, carefully remove upper aqueous layer without disturbing interface layer containing proteins.

9. Add 450 μL of methanol, vortex briefly.

10. Centrifuge for 5 minutes at 13,000-20,000 x g to pellet protein. Carefully remove and discard supernatant.

11. Open the lid to microfuge tube and allow protein pellet to air dry. Do not overdry the pellet!