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AAT Bioquest

FastClick™ Biotin Azide

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The reaction (Green Bar) of FastClick Cy5 Azide with coumarin alkyne occurs under extremely mild conditions (e.g., [Azide] = 0.02 mM, [Alkyne] = 0.02 mM, [CuSO4] = 0.02 mM, [Sodium Ascorbate] = 5 mM, in 100 mM HEPES) under which the common Cy5 azide does not effectively react with the coumarin alkyne substrate.
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Physical properties
Molecular weight456.57
SolventDMSO
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
Alternative formats
FastClick™ Biotin Alkyne
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OverviewpdfSDSpdfProtocol


See also: Click Chemistry
Molecular weight
456.57
FastClick™ Biotin Azide contains both the CAG moiety of FastClick (for assisting click efficiency) and biotin hapten (as the detection tag) for developing biotin-based probes. It readily reacts with an alkyne-containing biomolecule under extremely mild conditions. Biotin is one of the most commonly used tags that finds a variety of utilities in biological detections similarly to other popular haptens such as 2,4-Dinitrophenol (DNP) and DIG etc. Biotin conjugates are widely used in fluorescence imaging, fluorescence in situ hybridization (FISH) and nucleic acid detections in combination with the fluorescence-labeled streptavidin, avidin or NeutrAvidin® or other biotin-binding protein conjugates. FastClick™ reagents have been developed by the scientists of AAT Bioquest for enhancing the yield and reaction speed of copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. They contain a copper-chelating ligand that significantly stabilizes the Cu(I) oxidation state and thus accelerates the click reaction. They do not require the use of an external copper-chelator (such as the common THPTA or BTTAA). The high concentration of copper chelators is known to have a detrimental effect on DNA/RNA, thus causing biocompatibility issues. The introduction of a copper-chelating moiety at the reporter molecule allows for a dramatic raise of the effective Cu(I) concentration at the reaction site and thus accelerates the reaction. Under extremely mild conditions the FastClick™ azides and alkynes react much faster in high yield compared to the corresponding conventional CuAAC reactions.

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of FastClick™ Biotin Azide to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM219.024 µL1.095 mL2.19 mL10.951 mL21.902 mL
5 mM43.805 µL219.024 µL438.049 µL2.19 mL4.38 mL
10 mM21.902 µL109.512 µL219.024 µL1.095 mL2.19 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
FastClick™ Cy3 Azide55556915000010.1510.070.073
FastClick™ Cy5 Azide65167025000010.271, 0.420.020.03
FastClick™ Cy7 Azide7567792500000.30.050.036
FastClick™ XFD350 Azide34344119000-0.250.19
FastClick™ XFD488 Azide499520710000.9210.300.11
FastClick™ XFD555 Azide5535681500000.110.080.08
FastClick™ XFD647 Azide6506712390000.3310.000.03
FastClick™ XFD750 Azide7527762400000.1210.000.04

Images


References


View all 28 references: Citation Explorer
Rapid cell type-specific nascent proteome labeling in Drosophila.
Authors: Villalobos-Cantor, Stefanny and Barrett, Ruth M and Condon, Alec F and Arreola-Bustos, Alicia and Rodriguez, Kelsie M and Cohen, Michael S and Martin, Ian
Journal: eLife (2023)
Capture, Release, and Identification of Newly Synthesized Proteins for Improved Profiling of Functional Translatomes.
Authors: Phillips, Nancy J and Vinaithirthan, Bala M and Oses-Prieto, Juan A and Chalkley, Robert J and Burlingame, Alma L
Journal: Molecular & cellular proteomics : MCP (2023): 100497
Selective Removal of Unhydrolyzed Monolinked Peptides from Enriched Crosslinked Peptides To Improve the Coverage of Protein Complex Analysis.
Authors: An, Yuxin and Zhao, Qun and Gao, Hang and Zhao, Lili and Li, Xiao and Zhang, Xiaodan and Liang, Zhen and Zhang, Lihua and Zhang, Yukui
Journal: Analytical chemistry (2022): 3904-3913
Ultrastructural localization of de novo synthesized phosphatidylcholine in yeast cells by freeze-fracture electron microscopy.
Authors: Tsuji, Takuma and Fujimoto, Toyoshi
Journal: STAR protocols (2021): 100990
Profiling and Validation of Live-Cell Protein Methylation with Engineered Enzymes and Methionine Analogues.
Authors: Weiss, Nicole and Seneviranthe, Chamara and Jiang, Ming and Wang, Ke and Luo, Minkui
Journal: Current protocols (2021): e213
Maleimide-Based Chemical Proteomics for Quantitative Analysis of Cysteine Reactivity.
Authors: McConnell, Evan W and Smythers, Amanda L and Hicks, Leslie M
Journal: Journal of the American Society for Mass Spectrometry (2020)
Small Molecule Interactome Mapping by Photo-Affinity Labeling (SIM-PAL) to Identify Binding Sites of Small Molecules on a Proteome-Wide Scale.
Authors: Flaxman, Hope A and Miyamoto, David K and Woo, Christina M
Journal: Current protocols in chemical biology (2019): e75
Evaluation of click chemistry microarrays for immunosensing of alpha-fetoprotein (AFP).
Authors: Dadfar, Seyed Mohammad Mahdi and Sekula-Neuner, Sylwia and Trouillet, Vanessa and Liu, Hui-Yu and Kumar, Ravi and Powell, Annie K and Hirtz, Michael
Journal: Beilstein journal of nanotechnology (2019): 2505-2515
Lipopolysaccharide Upregulates Palmitoylated Enzymes of the Phosphatidylinositol Cycle: An Insight from Proteomic Studies.
Authors: Sobocińska, Justyna and Roszczenko-Jasińska, Paula and Zaręba-Kozioł, Monika and Hromada-Judycka, Aneta and Matveichuk, Orest V and Traczyk, Gabriela and Łukasiuk, Katarzyna and Kwiatkowska, Katarzyna
Journal: Molecular & cellular proteomics : MCP (2018): 233-254
Single-Protein-Specific Redox Targeting in Live Mammalian Cells and C. elegans.
Authors: Van Hall-Beauvais, Alexandra and Zhao, Yi and Urul, Daniel A and Long, Marcus J C and Aye, Yimon
Journal: Current protocols in chemical biology (2018): e43