Cy3 Azide Plus

Click Reaction Protocol for Staining Fixed/Permeabilized Cell

This is a general protocol for fixed/permeabilized cell imaging through a copper-catalyzed click reaction using the fluorescent Azide Plus reagent. We recommend using this protocol as a starting point for optimization of particular click chemistry procedures. We have found that a 1.5-3.0 μM concentration of Azide Plus reagent was optimal for most applications, including imaging of EdU incorporated into newly synthesized DNA and imaging of OPP labeled proteins without causing a high background signal. The optimal final concentration of the Azide Plus reagent is sample dependent and may range from 0.5 μM to 10 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:
   — 50 mM copper sulfate in water
   — 300 mM sodium ascorbate in water (dissolve 60 mg of sodium ascorbate in 1 mL of water)
   — Subtext
2. 1 mM Azide Plus reagent in DMSO or water
   
   

   Table 1

   Reac­tion Compo­nent	Number of cover­slipsor wells of a 96-well plate
   	1 cover­slip or 10 wells	5 cover­slips or 50 wells	10 cover­slips or 100 wells	20 cover­slips or 200 wells
   Reaction Buffer (Tris)	888 µL	4.4 mL	8.9 mL	17.8 mL
   50 mM Copper Sulfate	10 µL	50 µL	100 µL	200 µL
   AZDye Azide Plus­ Solu­tion (2 µM final concentration)	2 µL	10 µL	20 µL	40 µL
   Sodium ascorbate	100 µL	500 µL	1 mL	2 mL
   Total Volume	1 mL	5 mL	10 mL	20 mL

   
   
3. Remove the permeabilization buffer (if used). Wash the cells in each well twice with 1 ml of PBS. Remove PBS.
4. Immediately add 1 mL of the Reaction Cocktail to the sample. Evenly distribute the reaction cocktail over the sample.
5. Protect from light, and incubate the plate for 30 minutes at room temperature.
6. 6Remove the reaction cocktail. Wash each well once with 1 ml of Wash Buffer. Remove the Wash Buffer.
7. Wash each well with 1 mL of PBS. Remove PBS.

Click Reaction Protocol for Cell Lysates Labeling

This is a general protocol for labeling proteins in cell lysate through a copper-catalyzed click reaction using the fluorescent Azide Plus reagent. We recommend using this protocol as a starting point for optimization of particular click chemistry procedures. We have found that a 20 μM concentration of Azide Plus reagent was sufficient to label all alkyne-tagged proteins in the cell lysate without causing a high background signal. The optimal final concentration of the 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 Azide Plus reagent in DMSO or water
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 Azide Plus reagent stock solution (5 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.
8. Centrifuge for 10 minutes at 13,000-20,000 x g, then carefully remove upper aqueous layer without disturbing the interface layer containing proteins.
9. Add 450 µL of methanol, vortex briefly.
10. Centrifuge for 5 minutes at 13,000-20,000 × 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 over dry the pellet!