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ReadiLeave™ Reversible Biotin Succinimidyl Ester

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Physical properties
Molecular weight685.83
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


Molecular weight
685.83
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. ReadiLeave™ Reversible Biotin Succinimidyl Ester is an excellent building block to develop reversible biotin probes and products for biological detections and purification. 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

Prepare Protein Solution
  1. Prepare a 900 uL protein solution in 1X phosphate-buffered saline (PBS), pH 7.2-7.4.

    Note: If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4.

    Note: Protein solution should be free of stabilizers like bovine serum albumin (BSA) or gelatin.

    Note: The presence of sodium azide or thimerosal might also interfere with the conjugation reaction.

    Note: The protein concentration range of 2-10 mg/mL is recommended for optimal labeling efficiency.

  2. Add 100 µL of a reaction buffer (e.g., 1 M sodium bicarbonate solution or 1 M phosphate buffer with pH ~8.5 to 9.0) to the target protein solution to adjust pH to 8.5 ± 0.5.

Prepare ReadiLeave™ Reversible (RLR) Biotin Succinimidyl Ester Stock Solution
  1. Add anhydrous DMSO into the vial of RLR Biotin SE to make a 10 mM (6.85mg/ml) stock solution.

    Note: Prepare the dye stock solution before starting the conjugation. Use promptly.

    Note: RLR Biotin SE stock solution can be stored in the freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.

    Note: Extended storage of the dye stock solution may reduce the dye activity.

SAMPLE EXPERIMENTAL PROTOCOL

Run Conjugation Reaction

This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with RLR Biotin SE.

  1. Use a 10:1 molar ratio of RLR Biotin SE:Protein.

  2. Continue to rotate the reaction mixture at room temperature for 30-60 minutes.
Purify the Conjugate
  1. Purify the conjugate mixture to 1x PBS buffer (pH=7.2-7.4) with a ReadiUse™ Disposable PD-10 Desalting Column (Cat no. 60504) according to the manufacturer's instruction.
Measure Protein Concentration
  1. Protein concentration can be determined from the extinction coefficient by measuring absorbance at 280 nm.
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 the sample containing the target protein to the resin from the above section.

  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 Succinimidyl Ester 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 mM145.809 µL729.044 µL1.458 mL7.29 mL14.581 mL
5 mM29.162 µL145.809 µL291.617 µL1.458 mL2.916 mL
10 mM14.581 µL72.904 µL145.809 µL729.044 µL1.458 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 50 references: Citation Explorer
A multiple signal amplification photoelectrochemical biosensor based on biotin-avidin system for kanamycin sensing in fish and milk via synergism of g-C3N4 and Ru@SiO2.
Authors: Yao, Jun
Journal: Analytica chimica acta (2024): 342141
A Rapid and Simplified Method to Isolate Specific Regulators Based on Biotin-Avidin Binding Affinities in Crops.
Authors: Qin, Xiaojian and Li, Yuntong and Li, Cuiping and Li, Xiaowei and Wu, Yuanyuan and Wu, Qian and Wen, Huan and Jiang, Dan and Liu, Shifeng and Nan, Wenbin and Liang, Yongshu and Zhang, Hanma
Journal: Journal of agricultural and food chemistry (2024): 883-893
Preliminary efficacy of [90Y]DOTA-biotin-avidin radiotherapy against non-muscle invasive bladder cancer.
Authors: Alì, Alessandra and Leibowitz, Dev and Bhatt, Nikunj and Doubrovin, Mikhail and Spina, Catherine S and Bates-Pappas, Gleneara E and Taub, Robert N and McKiernan, James M and Mintz, Akiva and Molotkov, Andrei
Journal: European journal of nuclear medicine and molecular imaging (2023): 692-700
Biotinylated Quinone as a Chemiluminescence Sensor for Biotin-Avidin Interaction and Biotin Detection Application.
Authors: Kaladari, Fatema and El-Maghrabey, Mahmoud and Kawazato, Megumi and Kishikawa, Naoya and Kuroda, Naotaka
Journal: Sensors (Basel, Switzerland) (2023)
Biotin-Avidin System-Based Delivery Enhances the Therapeutic Performance of MSC-Derived Exosomes.
Authors: Deng, Daokun and Li, Xuan and Zhang, Jiu-Jiu and Yin, Yuan and Tian, Yi and Gan, Dian and Wu, Ruixin and Wang, Jia and Tian, Bei-Min and Chen, Fa-Ming and He, Xiao-Tao
Journal: ACS nano (2023): 8530-8550
Intraprocedural endothelial cell seeding of arterial stents via biotin/avidin targeting mitigates in-stent restenosis.
Authors: Alferiev, Ivan S and Hooshdaran, Bahman and Pressly, Benjamin B and Zoltick, Philip W and Stachelek, Stanley J and Chorny, Michael and Levy, Robert J and Fishbein, Ilia
Journal: Scientific reports (2022): 19212
A biotin-avidin-system-based virus-mimicking nanovaccine for tumor immunotherapy.
Authors: Lu, Zhuoxuan and Zhang, Yanwei and Wang, Yi and Tan, Guang-Hong and Huang, Feng-Ying and Cao, Rong and He, Nongyue and Zhang, Liming
Journal: Journal of controlled release : official journal of the Controlled Release Society (2021): 245-259
The Biotin-Avidin Interaction in Biotinylated Gold Nanoparticles and the Modulation of Their Aggregation.
Authors: Lyu, Yanchao and Martínez, Álvaro and D'Incà, Federica and Mancin, Fabrizio and Scrimin, Paolo
Journal: Nanomaterials (Basel, Switzerland) (2021)
Imparting Immunomodulatory Activity to Scaffolds via Biotin-Avidin Interactions.
Authors: Lurier, Emily B and Nash, Victoria A and Abee, Hannah S and Wissing, Tamar B and Bouten, Carlijn V C and Smits, Anthal I P M and Spiller, Kara L
Journal: ACS biomaterials science & engineering (2021): 5611-5621
Two chromatographic schemes for protein purification involving the biotin/avidin interaction under native conditions.
Authors: Raducanu, Vlad-Stefan and Tehseen, Muhammad and Shirbini, Afnan and Raducanu, Daniela-Violeta and Hamdan, Samir M
Journal: Journal of chromatography. A (2020): 461051