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The CuAAC Cell Reaction Buffer Kit (THPTA based) is suitable to perform Copper (Cu(I))-catalyzed Azide-Alkyne Click chemistry reactions (CuAAC) with cells containing metabolically functionalized Alkyne- or Azide modified biomolecules.
1 Kit provides sufficient amounts to perform 50 CuAAC experiments à 500 μl using 2 mM CuSO4 (copper source), 10 mM THPTA (Cu(I)-stabilizing ligand) and 100 mM Na-Ascorbate (reduction reagent) in 100 mM Na-Phosphate reaction buffer.
2 x 10 mg CuSO4 (M = 159.6 g/mol), #CLK-MI004)
Cu(I) stabilizing ligand:
5 x 25 mg THPTA (M= 434.5 g/mol, #CLK-1010)
4 x 200 mg Na-Ascorbate (M = 198.1 g/mol, #CLK-MI005)
2 x 30 ml sterile 100 mM Na-Phosphate Buffer, pH 7
10 ml sterile ddH2O
Materials required but not provided:
Alkyne-or Azide-functionalized substrates e.g. fixed and permeabilized cells containing metabolically functionalized Alkyne- or Azide-modified biomolecules.
(Picolyl)-Azide or Alkyne detection reagent and appropriate solvent (e.g. DMSO)
For labeling of fixed and permeabilized cells:
Washing solutions e.g. PBS containing 3% BSA
Fixation solution e.g. PBS containing 3.7% formaldehyd
Permeabilization solution e.g. PBS containing 0.5% Triton X-100
Mounting medium for imaging
Additional labeling reagent such as nuclear stain or antibody
Copper (Cu(I))-catalyzed Azide-Alkyne Click chemistry reactions (CuAAC) describe the reaction of an Azide-functionalized molecule A with a terminal Alkyne-functionalized molecule B that results in a stable conjugate A-B via a Triazole moiety.
Since terminal Alkynes are fairly unreactive towards Azides, the efficiency of CuAAC reactions strongly depends on the presence of a metal catalyst such as copper ions in the +1 oxidation state (Cu(I)).
Different copper sources, reduction reagents and Cu(I) stabilizing ligands are available however, for most bioconjugation applications the combination of the Cu(II) salt CuSO4 as copper source, a water-soluble Cu(I) stabilizing ligand such as BTTAA and sodium ascorbate as a reduction reagent is recommended.[1-3]
The use of Picolyl-Azide reagents instead of conventional Azide reagents can further increase the reaction efficiency and decrease the required final CuSO4 concentration due to the internal copper chelating moiety.
The set-up of a CuAAC reaction is based on the following general three-step procedure:
The CuAAC Cell Reaction Buffer Kit (THPTA based) provides sufficient amounts to perform 50 CuAAC experiments à 500 μl using 2 mM CuSO4, 10 mM THPTA and 100 mM Na-Ascorbate in 100 mM Na-Phosphate reaction buffer.
A general protocol for labeling of fixed and permeabilized cells containing metabolically functionalized Alkyne- or Azide-modified biomolecules is outlined below (see 3.) however, individual optimization might be required for different CUAAC labeling experiments as well as for critical reaction parameter e.g. final CuSO4 concentration, CuSO4:THPTA ratio, detection reagent concentration.
Hong et al. and Presolski et al. provide useful background information on the influence of CuSO4 concentration, CuSO4: ligand ratio and reaction buffer type that may be used as a starting point if optimization is required.
2. Preparation of stock solutions
The concentration of stock solutions (2.1 to 2.3) is suitable to prepare 500 μl assays containing 2 mM CuSO4, 10 mM THPTA and 100 mM Na-Ascorbate (see 3.3 and 3.4, respectively). Adjustments might be required if different assay volumes or final compound concentrations are used.
2.1 THPTA stock solution (Cu(I) stabilizing ligand)
Table 1 Volume of ddH2O required for a 250 mM THPTA stock solution.
|THPTA||Concentration of stock solution||Amount of ddH2O|
|25 mg||250 mM||230 μl|
Table 2 Volume of ddH2O required for a 100 mM CuSO4 stock solution.
|CuSO4||Concentration of stock solution||Amount of ddH2O|
|10 mg||100 mM||628 μl|
2.3 Na-Ascorbate stock solution (reduction reagent)
Please note: Do not use solutions that appear brown. Freshly prepared, fully functional Na-Ascorbate solutions are colorless and turn brown upon oxidization thereby losing their reduction capability.
Table 3 Volume of ddH2O required for a 1 M Na-Ascorbate stock solution.
|Na-Ascorbate||Concentration of stock solution||Amount of ddH2O|
|200 mg||1 M||1010 μl|
2.4 (Picolyl)-Azide detection reagent stock solution
3. General protocol for CLICK labeling of fixated and permeabilized cells containing metabolically functionalized Alkyne- or Azide-modified biomolecules
The protocol below is intended as a general guideline however, individual optimization might be required.
3.1 Metabolically label cells with an Alkyne or Azide-functionalized substrate
3.2 Fixate and permeabilize cells
Please note: The fixation with 3.7% formaldehyde in PBS and subsequent permeabilization with 0.5% Triton X-100 is a general guideline. Optimization might be required. Different reagent concentrations, different fixation and permeabilization reagents (e.g. methanol or saponin) or TBS as buffer solution intstead of PBS can be used as well. Permeabilization is not required for cell surface or lipid component labeling.
3.3 Prepare CuSO4:THPTA-Premix
Please note: Both the final CuSO4 concentration as well as CuSO4:THPTA ratio are critical parameters for CuAAC reaction efficiency. A final CuSO4 concentration of 2 mM and a CuSO4:THPTA ratio of 1:5 is recommended as a starting point for labeling of fixed and permeabilized cells containing metabolically Azide- or Alkyne-functionalized biomolecules. Individual optimization for each assay is strongly recommended. Minimum CuSO4 concentration: 50 μM.
Table 4 Pipetting scheme for CuSO4:THPTA-Premix (ratio 1:5).
|Compound||Final conc.||1 Assay||10 Assays|
|100 mM CuSO4 stock solution (see 2.2)||33.33 mM||10 μl||100 μl|
|250 mM THPTA stock solution (see 2.1)||166.66 mM||20 μl||200 μl|
3.4 Prepare CLICK reaction cocktail
Please note: Prepare CLICK reaction cocktail freshly for each experiment and use it immediately but definitely within 15 minutes after preparation. 500 μl CLICK reaction cocktail (1 Assay) is sufficient to label one 18x18 coverslip.
Table 5 Pipetting scheme for CLICK reaction cocktail. Please add the compounds exactly in the order described below.
|Compound||Final conc.||1 Assay (500 μl)||10 Assays (5 ml)|
|100 mM Na-Phosphate reaction buffer, pH 7||419 μl||4.19 ml|
|10 mM Azide or-Alkyne detection reagent stock solution (not provided, see 2.4)||20 μM||1 μl||10 μl|
|33.33 mM / 166.66 mM CuSO4:THPTA-Premix (see 3.3)||2 mM / 10 mM||30 μl||300 μl|
|1 M Na-Ascorbate stock solution (see 2.3)||100 mM||50 μl||500 μl|
3.5 Perform CLICK labeling of fixated and permeabilized Alkyne- or Azide-labeled cells
Please click the black arrow on the right to expand the citation list. Click publication title for the full text.
 Presolski et al. (2011) Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation. Current Protocols in Chemical Biology 3:153.
 Hong et al. (2011) Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation. Angew. Chem. Int. Ed. 48:9879.
 Besanceney-Webler et al. (2011) Increasing the Efficiacy of Bioorthogonal Click Reactions for Bioconjugation: A Comparative Study. Angew. Chem. Int. Ed. 50:8051.
 Uttamapinant et al. (2012) Fast, Cell-Compatible Click Chemistry with Copper-Chelating Azides for Biomolecular Labeling. Angew. Chem. Int. Ed. 51:5852.