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The CuAAC Biomolecule Reaction Buffer Kit (THPTA based) is suitable to perform Copper (Cu(I))-catalyzed Azide-Alkyne Click chemistry reactions (CuAAC) with Azide- or Alkyne- modified biomolecules.
1 Kit provides sufficient amounts to perform 25 CuAAC experiments à 200 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.
1 x 10 mg CuSO4 (M = 159.6 g/mol), #CLK-MI004)
Cu(I) stabilizing ligand:
1 x 25 mg THPTA (M= 434.5 g/mol, #CLK-1010)
1 x 200 mg Na-Ascorbate (M = 198.1 g/mol, #CLK-MI005)
1 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.
a) fixed and permeabilized cells containing metabolically functionalized Alkyne- or Azide-modified biomolecules.
b) cell lysate containing metabolically functionalized Alkyne- or Azide-modified proteins.
(Picolyl)-Azide or Alkyne detection reagent and appropriate solvent (e.g. DMSO)
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 THPTA 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 Biomolecule Reaction Buffer Kit (THPTA based) provides sufficient amounts to perform 25 CuAAC experiments à 200 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 biomolecules (see 3.) is outlined below. Individual optimization might however be required for different CUAAC labeling experiments as well as for critical reaction parameter e.g. final CuSO4 concentration, CuSO4:ligand 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
Please note: The concentration of stock solutions (2.1 to 2.3) is suitable to prepare 200 and 500 μl assays containing 2 mM CuSO4, 10 mM THPTA and 100 mM Na-Ascorbate (see 3.1 and 3.2, 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 biomolecules
The protocol below is intended as a general guideline however, individual optimization might be required.
The amount of provided reagents is sufficient to perform 25 CuAAC experiments à 200 μl using 2 mM CuSO4, 10 mM THPTA and 100 mM Na-Ascorbate in 100 mM Na-Phosphate reaction buffer.
3.1 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 Azide- and Alkyne-functionalized biomolecules with a correspondingly labeled detection reagent. 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|
|100 mM CuSO4 stock solution (see 2.2)||33.33 mM||4 μl|
|250 mM THPTA stock solution (see 2.1)||166.66 mM||8 μl|
3.2 Perform CLICK labeling
Please note: The protocol below describes CuAAC labeling of an Alkyne-functionalized biomolecule (e.g. cell lysate containing Alkyne-functionalized proteins) with an Azide-functionalized detection reagent (e.g. Azide-functionalized fluorescent dye). It can be used vice versa as well (Azide-functionalized biomolecule and Alkyne-functionalized detection reagent).
Table 5 Starting amount of Alkyne-functionalized biomolecules. Please note: The stated amounts are intended for an orientation only. They may need to be adjusted depending on the final read-out or downstream processing after CLICK reaction.
|Substrate||Final Amount||Recommended final assay volume|
|Cell lysate containing Alkyne-functionalized proteins||50 μg||200 μl|
|Single Alkyne-functionalized oligonucleotide||5 - 10 nmol||20-50 μl|
|Multiple Alkyne-functionalized DNA or RNA fragments generated by enzymatic incorporation of correspondingly labeled nucleotides||3 - 15 pmol*||20-50 μl|
Table 6 Pipetting scheme for a 200 μl CLICK reaction assay. Please add the compounds exactly in the order described below.
|Compound||Final conc./amount||1 Assay (200 μl)|
|Alkyne-functionalized biomolecule||see tab. 5||X μl|
|100 mM Na-Phosphate reaction buffer, pH 7||100 mM||ad 167 μl|
|10 mM Azide-functionalized detection reagent stock solution (not provided, see 2.4)||50 μM||1 μl|
|33.33 mM / 166.66 mM CuSO4:THPTA-Premix (see 3.1)||2 mM / 10 mM||12 μl|
|1 M Na-Ascorbate stock solution (see 2.3)||100 mM||20 μl|
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.