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CuAAC Biomolecule Reaction Buffer Kit (BTTAA based)

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CLK-071 1 kit 121,45 Add to Basket/Quote Add to Notepad

For research use only!

Shipping: shipped at ambient temperature

Storage Conditions: store at 4 °C
Short term exposure (up to 1 week cumulative) to ambient temperature possible.

Shelf Life: 12 months after date of delivery

Description:
The CuAAC Biomolecule Reaction Buffer Kit (BTTAA 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 BTTAA (Cu(I)-stabilizing ligand) and 100 mM Na-Ascorbate (reduction reagent) in 100 mM Na-Phosphate reaction buffer.

Content:
Copper source:
1 x 10 mg CuSO4 (M = 159.6 g/mol), #CLK-MI004)

Cu(I) stabilizing ligand:
1 x 25 mg BTTAA (M= 430.5 g/mol, #CLK-067)

Reduction Reagent:
1 x 200 mg Na-Ascorbate (M = 198.1 g/mol, #CLK-MI005)

Reaction Buffer:
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)

1. Introduction

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 or BTTAA and sodium ascorbate as a reduction reagent is recommended.[1-3] BTTAA promotes a higher reaction efficiency under some experimental conditions[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.[4] Especially the combination with BTTAA as ligand may allow you to use a decreased copper concentration while maintaining similar reaction efficiencies achieved with traditional Azide reagents.

The set-up of a CuAAC reaction is based on the following general three-step procedure:

  • Prepare a mix of Alkyne- and Azide functionalized molecules in an appropriate reaction buffer.
  • Prepare a CuSO4: Cu(I)-ligand premix, add it to the Azide- Alkyne solution and mix briefly.
  • Add Na-Ascorbate as reduction reagent at last to start the reaction.


The CuAAC Biomolecule Reaction Buffer Kit (BTTAA based) provides sufficient amounts to perform 25 CuAAC experiments à 200 μl using 2 mM CuSO4, 10 mM BTTAA 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.[2] and Presolski et al.[1] 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 BTTAA 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 BTTAA stock solution (Cu(I) stabilizing ligand)

  • Add an appropriate amount of ddH2O (Tab. 1), vortex until the compound is completely dissolved and spin down briefly.
  • Prepare aliquots to avoid repeated freeze-thaw-cycles and store at -20 °C.
  • The solution is stable up to 1 year at -20 °C.
  • BTTAA is less soluble than THPTA. If required, heat shortly up to 70 °C to achieve a clear solution.

Table 1 Volume of ddH2O required for a 50 mM BTTAA stock solution.

BTTAAConcentration of stock solutionAmount of ddH2O
25 mg50 mM1163 μl


2.2 CuSO4 stock solution (copper source)

  • Add an appropriate amount of ddH2O (Tab. 2), vortex until the compound is completely dissolved and spin down briefly.
  • Prepare aliquots to avoid repeated freeze-thaw-cycles and store at -20 °C.
  • The solution is stable up to 1 year at -20 °C.

Table 2 Volume of ddH2O required for a 100 mM CuSO4 stock solution.

CuSO4Concentration of stock solutionAmount of ddH2O
10 mg100 mM628 μl

2.3 Na-Ascorbate stock solution (reduction reagent)

  • Add an appropriate amount of ddH2O (Tab. 3), vortex until the compound is completely dissolved and spin down briefly.
  • Prepare aliquots to avoid repeated freeze-thaw-cycles and store at -20 °C.
  • The solution is stable up to 1 year at -20 °C.

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-AscorbateConcentration of stock solutionAmount of ddH2O
200 mg1 M1010 μl

2.4 (Picolyl)-Azide detection reagent stock solution

  • (Picolyl)-Azide detection reagents are not provided within this kit.
  • Add an appropriate amount of suitable solvent e.g. DMSO to achieve a stock solution concentration of 10 mM for Azide detection reagents and 500 μM for Picolyl-Azide detection reagents.
  • Final concentrations of Azide or Alkyne detection reagents may range from 2 μM to 100 μM. Final concentrations of Picolyl-Azide detection reagents may range from 0.5 to 5 μM (see 3.4).
  • If the molar amount of Alkyne-groups can be calculated, we recommend starting with a 10-fold molar excess of Azide-functionalized detection reagent.

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 BTTAA and 100 mM Na-Ascorbate in 100 mM Na-Phosphate reaction buffer.

3.1 Prepare CuSO4:BTTAA-Premix

Please note: Both the final CuSO4 concentration as well as CuSO4:BTTAA ratio are critical parameters for CuAAC reaction efficiency. A final CuSO4 concentration of 2 mM and a CuSO4:BTTAA 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.

  • Prepare the CuSO4:BTTAA-Premix freshly for each experiment.
  • Allow all solutions to warm up to room temperature.
  • Mix the appropriate amount of 100 mM CuSO4 and 50 mM BTTAA stock solution (Tab. 4) by vortexing and spin down briefly.
  • 44 μl CuSO4:BTTAA-Premix (1 Assay) is sufficient for the preparation of 500 μl CLICK reaction assay (see. 3.2).

Table 4 Pipetting scheme for CuSO4:BTTAA-Premix (ratio 1:5).

CompoundFinal conc.1 Assay
100 mM CuSO4 stock solution (see 2.2)9.1 mM4 μl
50 mM BTTAA stock solution (see 2.1)45.45 mM40 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).

  • Allow all solutions to warm up to room temperature.
  • Final assay volume: 200 μl.
  • Refer to Tab. 6 for appropriate amounts of stock solutions.
  • Mix an Alkyne-functionalized biomolecule (see Tab. 5) with an appropriate amount of 100 mM Na-Phosphate reaction buffer, pH 7 to achieve a final volume of 135 μl.



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.

SubstrateFinal AmountRecommended final assay volume
Cell lysate containing Alkyne-functionalized proteins50 μg200 μl
Single Alkyne-functionalized oligonucleotide5 - 10 nmol20-50 μl
Multiple Alkyne-functionalized DNA or RNA fragments generated by enzymatic incorporation of correspondingly labeled nucleotides3 - 15 pmol*20-50 μl

*e.g. 3 pmol correspond to 1.5 μg of a 1500 bp RNA fragment or 1 μg of a 500 bp DNA fragment.

  • Add 1 μl of a 10 mM Azide- functionalized detection reagent stock solution (see 2.4) vortex and spin-down briefly (final concentration: 50 μM). If the molar amount of Alkyne-groups can be calculated, we recommend starting with a 10-fold molar excess of Azide-functionalized detection reagent.
  • Add 44 μl CuSO4/BTTAA-Premix (see. 3.1), vortex and spin down briefly.
  • Add 20 μl 1 M Na-Ascorbate stock solution (see 2.3) to initiate the reaction, vortex and spin down briefly.
  • Incubate samples 30 – 60 min at room temperature or 37 °C (protected from light).
  • Alkyne-functionalized biomolecules are now CLICK-labeled and ready for downstream processing and/or analysis.



Table 6 Pipetting scheme for a 200 μl CLICK reaction assay. Please add the compounds exactly in the order described below.

CompoundFinal conc./amount1 Assay (200 μl)
Alkyne-functionalized biomoleculesee tab. 5X μl
100 mM Na-Phosphate reaction buffer, pH 7100 mMad 135 μl
10 mM Azide-functionalized detection reagent stock solution (not provided, see 2.4)50 μM[1]1 μl
9.1 mM / 45.45 mM CuSO4:BTTAA-Premix (see 3.1)2 mM / 10 mM44 μl
1 M Na-Ascorbate stock solution (see 2.3)100 mM20 μl

[1]Final concentrations of Azide or Alkyne detection reagents may range from 2 μM to 100 μM. Final concentrations of Picolyl-Azide detection reagents may range from 0.5 to 5 μM. We recommend starting with 50 μM or 5 μM, respectively in case the molar amount of Alkyne-groups is unknown. If the molar amount of Alkyne-groups can be calculated, we recommend starting with a 10-fold molar excess of Azide-functionalized detection reagent. Concentrations can be titrated down in case of high background or up in case of low signal.

Product Citations:
Please click the black arrow on the right to expand the citation list. Click publication title for the full text.

Selected References:
[1] Presolski et al. (2011) Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation. Current Protocols in Chemical Biology 3:153.
[2] Hong et al. (2011) Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation. Angew. Chem. Int. Ed. 48:9879.
[3] Besanceney-Webler et al. (2011) Increasing the Efficiacy of Bioorthogonal Click Reactions for Bioconjugation: A Comparative Study. Angew. Chem. Int. Ed. 50:8051.
[4] Uttamapinant et al. (2012) Fast, Cell-Compatible Click Chemistry with Copper-Chelating Azides for Biomolecular Labeling. Angew. Chem. Int. Ed. 51:5852.