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For general laboratory use.
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 (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.
Content:
Copper source:
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)Reduction Reagent:
1 x 200 mg Na-Ascorbate (M = 198.1 g/mol, #CLK-MI005)Reaction Buffer:
1 x 30 ml 100 mM Na-Phosphate Buffer, pH 710 ml 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 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.[4]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 (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.[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 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)
- 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.
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 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.
CuSO4 Concentration of stock solution Amount of ddH2O 10 mg 100 mM 628 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 to slightly yellow 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
- (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 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.
- Prepare the CuSO4:THPTA-Premix freshly for each experiment.
- Allow all solutions to warm up to room temperature.
- Mix the appropriate amount of 100 mM CuSO4 and 250 mM THPTA stock solution (Tab. 4) by vortexing and spin down briefly.
- 12 μl CuSO4:THPTA-Premix (1 Assay) is sufficient for the preparation of 500 μl CLICK reaction assay (see. 3.2).
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).
- 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 167 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.
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 *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 12 μl CuSO4/THPTA-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.
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] 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 [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.
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产品组分
内容 型号
规格 储存温度
CuAAC生物分子反应缓冲液试剂盒(THPTA)
CLK072-01 1set
-20°C CuAAC生物分子反应缓冲液试剂盒(THPTA)
CLK072-02 2set
-20°C 操作手册
1 1 常温
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注意事项
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FAQ
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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.Patent Oligonucleotide-tethered nucleotides
Arvydas Lubys, Inga Cikotiene, ..., Sihong Chen
us11739316 | 2023 Aug 29
Detection of base analogs incorporated during DNA replication by nanopore sequencing
Daniela Georgieva, Qian Liu, ..., Dieter Egli
Nucleic Acids Research | 2020 Sep 04 | 32710620
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