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ReadiLeave™ Reversible Biotin Azide

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Physical properties
Molecular weight615.79
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
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OverviewpdfSDSpdfProtocol


See also: Click Chemistry
Molecular weight
615.79
ReadiLeave™ Reversible (RLR) Biotin is a newly developed biotin derivative that has significantly reduced affinity to avidin (including streptavidin) to make the binding of RLR biotin and streptavidin readily reversible when needed. It is complimentary to the regular biotin and has a moderate affinity to streptavidin to ensure a tight binding but not too tight to be reversed in contrast with the regular non-reversible biotin. ReadiLeave™ Reversible Biotin Azide is an excellent building block to develop reversible biotin probes and products for biological detections and purification using the well-known click reactions (CuAAC). It readily reacts with an alkyne-modified biomolecule under mild conditions. The affinity between streptavidin and biotin might be the strongest non-covalent interactions known in biological interactions. Streptavidin, a homotetrameric protein, exhibits an extraordinarily high affinity for biotin. Each streptavidin monomer can bind one biotin molecule, allowing a streptavidin protein to maximally bind four biotins. The streptavidin-biotin interaction is highly specific and remains robust under a wide range of conditions. Biotin can readily be attached to proteins, nucleic acids, or even nanoparticles. Once formed, the bond between biotin and streptavidin is unaffected by extremes of pH, temperature, organic solvents, and other denaturing agents. This powerful interaction has been exploited for various applications such as ELISA, Western blotting, Northern blotting, Southern blotting, immunohistochemistry (IHC), cell surface labeling, Fluorescence-Activated Cell Sorting (FACS), and electrophoretic Mobility Shift Assays (EMSA) etc.

Example protocol


PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Required Stock Solutions for Labeling Alkyne-Modified Biomolecules
  1. 200 mM THPTA [tris (3-hydroxypropyltriazolylmethyl) amine)] ligand in water
  2. 100 mM CuSO4 in water

  3. 100 mM sodium ascorbate in water
  4. 10 mM ReadiLeave™ Reversible (RLR) Biotin Azide in DMSO

Required Stock Solutions for Labeling Cells, Cell Lysates, or Biologial Samples
  1. 100 mM THPTA [tris (3-hydroxypropyltriazolylmethyl) amine)] ligand in aqueous buffer or water
  2. 20 mM CuSO4 in water
  3. 300 mM sodium ascorbate in water
  4. 5 mM ReadiLeave™ Reversible (RLR) Biotin Azide in DMSO

SAMPLE EXPERIMENTAL PROTOCOL

Labeling Oligonucleotides with RLR Boitin Azide
  1. Prepare the required stock solutions for labeling alkyne-modified biomolecules from the 'Preparation of Stock Solutions' section above.

  2. Prepare an alkyne-modified oligo in water as concentrated as possible (e.g., >10 mg/mL).
  3. Mix and vortex well CuSO4 with THPTA in a 1:2 ratio for several minutes before the reaction. This working solution is stable for several weeks when frozen.
  4. To the alkyne-modified oligo solution, add an excess of RLR Biotin Azide (2-5 equivalents by molar ratio).
  5. Add 5 equivalents of THPTA/CuSO4 working solution (from Step 1).
  6. Add 10-30 equivalents of sodium ascorbate.
  7. Stir, vortex or shake the reaction mixture at room temperature for 30-60 minutes.
  8. Ethanol-precipitate the oligo or purify it using your desired method (e.g., HPLC).
Labeling Peptides with RLR Biotin Azide
  1. Prepare the required stock solutions for labeling alkyne-modified biomolecules from the 'Preparation of Stock Solutions' section above.
  2. Prepare an alkyne-modified peptide in water of DMF as concentrated as possible (e.g., >10 mg/mL).
  3. Incubate CuSO4 with THPTA ligand in a 1:2 ratio several minutes before the reaction. This solution is stable for several weeks when frozen.
  4. To the alkyne-modified peptide solution, add an excess of RLR Biotin Azide (5-10 equivalents by molar ratio).
  5. Add 5-10 equivalents of THPTA/CuSO4.
  6. Add 10-20 equivalents of sodium ascorbate.
  7. Stir, vortex, or shake the reaction mixture at room temperature for 30-60 minutes.
  8. Purify your desired peptide by HPLC.
Labeling Small Organic Alkyne Molecules with RLR Biotin Azide
  1. Prepare the required stock solutions for labeling alkyne-modified biomolecules from the 'Preparation of Stock Solutions' section above.
  2. Prepare an alkyne compound in water or DMF as concentrated as possible (e.g., >10 mg/mL).
  3. Incubate CuSO4 with THPTA ligand in a 1:2 ratio several minutes before the reaction. This solution is stable for several weeks when frozen.
  4. To the alkyne solution, add an excess of RLR Biotin Azide (5-10 equivalents by molar ratio).
  5. Add 25 equivalents of THPTA/CuSO4.
  6. Add 50 equivalents of sodium ascorbate.
  7. Stir the reaction mixture at room temperature for 30-60 minutes.
  8. Purify your desired molecule by chromatography or other methods.
Labeling Biopolymers with RLR Biotin Azide
  1. Prepare the required stock solutions for labeling alkyne-modified biomolecules from the 'Preparation of Stock Solutions' section above.
  2. Prepare an alkyne-modified biopolymer in water as concentrated as possible (e.g., >10 mg/mL).
  3. Incubate CuSO4 with THPTA ligand in a 1:2 ratio several minutes before the reaction. This solution is stable for several weeks when frozen.
  4. To the alkyne-modified biopolymer solution, add an excess of RLR Biotin Azide (Loading ratio: 5-20 RLR Botin azide).
  5. Add 5 molar equivalents (referenced to RLR Biotin Azide) of THPTA/CuSO4.
  6. Add 10 equivalents of sodium ascorbate (referenced to RLR Biotin Azide).
  7. Stir, vortex or shake the reaction mixture at room temperature for 30-60 minutes.
  8. Purify your desired molecule by gel filtration or dialysis.
Labeling Cells, Cell Lysates or Biological Samples with RLR Biotin Azide
  1. Prepare the required stock solutions for labeling cells, cell lysates, or biological samples from the 'Preparation of Stock Solutions' section above.
  2. For each alkyne-modified cell or cell lysate sample, add the following reagents to a 1.5 mL microfuge tube, then vortex briefly to mix:

    • 50 µL cell or cell lysate sample
    • 50 µL PBS buffer
    • 50 µL of 5 mM RLR Biotin Azide in DMSO or water
  3. Add 10 µL of 100 mM THPTA solution, and vortex briefly to mix.
  4. Add 10 µL of 20 mM CuSO4 solution and vortex briefly to mix.
  5. Add 10 µL of 300 mM sodium ascorbate solution to initiate a click reaction, and vortex briefly to mix.
  6. Protect reaction from light and allow click reaction to incubate for 30 minutes at room temperature.
  7. Cells or cell lysates are now click-labeled and ready for downstream processing and/or analysis.
Protocol for Target Protein Pull-down Assays

Section 1: Coupling RLR Biotinylated Protein to a Resin

  1. Select a streptavidin-resin suitable for your application.
  2. Wash and equilibrate the resin by adding 1xPBS or a suitable wash buffer.
  3. Add appropriate amounts of RLR Biotinylated protein and incubate for 30 minutes.
  4. Wash the resin to remove unlabeled protein and equilibrate with PBS.

Section 2: Pull-down the Target Protein

  1. Add a sample containing the target protein to the resin from the Section 1.
  2. Incubate for 60 minutes.
  3. The target protein will be pulled down by RLR Biotinylated protein resin from Section 1.

Section 3: Elution of the Target Protein

  1. Centrifuge the resin to remove the supernatant and wash the resin by adding 1xPBS buffer (pH=7.2~7.4) or a suitable wash buffer.
  2. Repeat washing as needed.
  3. Add elution buffer (4 mM d-biotin in 20 mM Tris-HCl Buffer (pH=7.5) with 50 mM NaCl) and incubate at 37°C for 10 minutes or longer. Repeat three times or as needed.
  4. Pool all the elution, and the target protein and RLR biotinylated protein complex will be ready for further analysis.

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of ReadiLeave™ Reversible 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 mM162.393 µL811.965 µL1.624 mL8.12 mL16.239 mL
5 mM32.479 µL162.393 µL324.786 µL1.624 mL3.248 mL
10 mM16.239 µL81.197 µL162.393 µL811.965 µL1.624 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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Images


References


View all 29 references: Citation Explorer
An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation.
Authors: Rajkumar, Shreenidhi and Dixon, Danielle and Lipchik, Andrew M and Gruber, Joshua J
Journal: Journal of visualized experiments : JoVE (2024)
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