ReadiLeave™ Reversible Biotin Alkyne

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

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 Alkyne in DMSO

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 Alkyne in DMSO

1. Prepare the required stock solutions for labeling azide-modified biomolecules from the 'Preparation of Stock Solutions' section above.
2. Prepare an azide-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 azide-modified oligo solution, add an excess of RLR Biotin Alkyne (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).

1. Prepare the required stock solutions for labeling azide-modified biomolecules from the 'Preparation of Stock Solutions' section above.
2. Prepare an azide-modified peptide 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 azide-modified peptide solution, add an excess of RLR Biotin Alkyne (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.

1. Prepare the required stock solutions for labeling azide-modified biomolecules from the 'Preparation of Stock Solutions' section above.
2. Prepare an azide 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 azide solution, add an excess of RLR Biotin Alkyne (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.

1. Prepare the required stock solutions for labeling azide-modified biomolecules from the 'Preparation of Stock Solutions' section above.
2. Prepare an azide-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 azide-modified biopolymer solution, add an excess of RLR Biotin Alkyne (Loading ratio: 5-20 RLR Botin Alkyne).
5. Add 5 molar equivalents (referenced to RLR Biotin Alkyne) of THPTA/CuSO4.
6. Add 10 equivalents of sodium ascorbate (referenced to RLR Biotin Alkyne).
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.

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 azide-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 Alkyne 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.

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.