logo
AAT Bioquest

ReadiLeave™ Reversible Biotin Maleimide

Product Image
Product Image
Gallery Image 1
Ordering information
Price
Catalog Number
Unit Size
Quantity
Add to cart
Additional ordering information
Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
ShippingStandard overnight for United States, inquire for international
Request quotation
Physical properties
Molecular weight625.78
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
Related products
Biotin *CAS 58-85-5*
Biotin, succinimidyl ester *CAS 35013-72-0*
Biotin ethylenediamine *CAS 1217450-40-2*
Biotin cadaverine
Biotin C2 maleimide
Biotin-4-fluorescein *CAS 1032732-74-3*
Biotin hydrazide *CAS 66640-86-6*
Biotin-X NTA [Biotin-X nitrilotriacetic acid, potassium salt] *CAS 856661-92-2*
Biotin-X, succinimidyl ester *CAS 72040-63-2*
Biotin PEG2 amine *CAS 138529-46-1*
Biotin PEG2 maleimide *CAS 305372-39-8*
Biotin PEG2 succinimidyl ester
Biotin-PEG3-azide *CAS 875770-34-6*
Biotin Azide
Biotin Alkyne *CAS 773888-45-2*
Biotin PEG4 succinimidyl ester
ReadiView™ biotin acid
ReadiView™ biotin amine
ReadiView™ biotin hydrazide
ReadiView™ biotin maleimide
ReadiView™ biotin succinimidyl ester
ReadiLink™ Protein Biotinylation Kit *Powered by ReadiView™ Biotin Visionization Technology*
Amplite® Colorimetric Biotin Quantitation Kit
Biotin-11-dUTP *1 mM in TE Buffer (pH 7.5)* *CAS 86303-25-5*
Biotin-16-dUTP *1 mM in TE Buffer (pH 7.5)* *CAS 136632-31-0*
Biotin-20-dUTP *1 mM in TE Buffer (pH 7.5)*
Cal-520®-Biotin Conjugate
Phalloidin-Biotin Conjugate
FMOC-Lys(Biotin)-OH
Biotin-14-dCTP *1 mM in Tris Buffer (pH 7.5)*
Biotin PEG3 amine
Annexin V-Biotin conjugate
N6-Methyladenosine-Biotin conjugate
Biotin Styramide *Superior Replacement for Biotin Tyramide*
Cy5 biotin conjugate
Biotin-cAMP conjugate
2',3'-cGAMP-Biotin conjugate
Cy5.5 biotin conjugate
Biotin-PEG4-tyramide
HRP-Biotin Conjugate
Biotin-11-dATP *1 mM in Tris Buffer (pH 7.5)*
Biotin-11-dGTP *1 mM in Tris Buffer (pH 7.5)*
Biotin NTA
Biotin-X IDA
ReadiCleave™ SSL biotin NHS ester
Biotin-dT Phosphoramidite
ReadiLink™ Biotin Nick Translation dsDNA Labeling Kit
ReadiLink™ Biotin Oligo and ssDNA Labeling Kit
AF532 PEG4 biotin conjugate
Biotin PEG 4 Alkyne
Cy3 biotin conjugate
Cy7 biotin conjugate
Biotin C2 Azide
mFluor™ UV 375 Biotin Conjugate
mFluor™ UV 460 Biotin Conjugate
mFluor™ Violet 500 Biotin Conjugate
mFluor™ Violet 540 Biotin Conjugate
mFluor™ Red 780 Biotin Conjugate
Biotin Phosphoramidite
Bio-16-UTP [Biotin-16-UTP] *1 mM*
Biotin PEG4 amine
mFluor™ Violet 450-PEG4-Biotin Conjugate
Acridinium Biotin Conjugate
FastClick™ Biotin Azide
FastClick™ Biotin Alkyne
Xylazine-Biotin Conjugate
Amplite® Rapid Colorimetric Biotin Quantitation Kit *Optimized to Use with Nanodrop*
Amplite® Fluorimetric Biotin Quantitation Kit
Portelite™ Rapid Fluorimetric Biotin Quantitation Kit *Optimized to Use with CytoCite™ and Qubit™ Fluorometers*
Biotin-PEG2-azide *CAS 945633-30-7*
Show More (60)

OverviewpdfSDSpdfProtocol


Molecular weight
625.78
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 Maleimide is an excellent building block to develop reversible biotin probes and products for biological detections and purification. It readily reacts with a thiol-containing molecule under neutral to slightly acidic conditions with high yield. 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: The pH of the protein solution should be 6.5 ± 0.5.

    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.

Optional: Disulfide Reduction

If your protein does not contain a free cysteine, you must treat your protein with DTT or TCEP to generate a thiol group. DTT or TCEP converts disulfide bonds to two free thiol groups. If you use DTT, you must remove free DTT by dialysis or gel filtration before conjugating the ReadiLeave™ Reversible Biotin Maleimide to your protein. The following is a sample protocol for generating a free thiol group:

  1. Prepare a fresh solution of 1 M DTT (15.4 mg/100 µL) in distilled water.

  2. To make an IgG solution in 20 mM DTT, add 20 µL of DTT solution per 1 mL of IgG solution while mixing well. Allow the solution to stand at room temperature for 30 minutes without additional mixing to reduce the oxidation of cysteines to cystines.

  3. Pass the reduced IgG through the filtration column that has been pre-equilibrated with "Exchange Buffer." Collect 0.25 mL fractions from the column.

  4. Determine the protein concentrations and pool the fractions with the majority of the IgG. This can be done either spectrophotometrically or colorimetrically.

  5. It is recommended to carry out the conjugation immediately after this step. Please refer to the Sample Experiment Protocol for more details.

    Note: IgG solutions should be >4 mg/mL for the best results. The protein should be concentrated if less than 2 mg/mL. An additional 10% should be included for losses on the buffer exchange column.

    Note: The reduction can be carried out in almost any buffers from pH 7-7.5 (e.g., MES, phosphate, or TRIS buffers).

    Note: Steps 3 and 4 can be replaced by dialysis.

Prepare ReadiLeave™ Reversible (RLR) Biotin Maleimide Stock Solution
  1. Add anhydrous DMSO into the vial of RLR Biotin maleimide to make a 10 mM (6.85 mg/mL) stock solution.  Mix well by pipetting or vortexing.

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

    Note: RLR Biotin maleimide 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 Maleimide.

  1. Use a 10:1 molar ratio of RLR Biotin Maleimide: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 Section 1 above.

  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 Maleimide 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 mM159.801 µL799.003 µL1.598 mL7.99 mL15.98 mL
5 mM31.96 µL159.801 µL319.601 µL1.598 mL3.196 mL
10 mM15.98 µL79.9 µL159.801 µL799.003 µL1.598 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Images


References


View all 50 references: Citation Explorer
Highly Sensitive, Stable InP Quantum Dot Fluorescent Probes for Quantitative Immunoassay Through Nanostructure Tailoring and Biotin-Streptavidin Coupling.
Authors: Zhang, Lifang and Xu, Han and Zhang, Xuhui and Chen, Xinxin and Lv, Yanbing and Zhang, Ruixue and Wang, Lei and Wu, Ruili and Shen, Huaibin and Li, Lin Song
Journal: Inorganic chemistry (2024)
An artificial nickel chlorinase based on the biotin-streptavidin technology.
Authors: Yu, Kun and Zhang, Kailin and Jakob, Roman P and Maier, Timm and Ward, Thomas R
Journal: Chemical communications (Cambridge, England) (2024): 1944-1947
Comparative study on different immobilization sites of immobilized β-agarase based on the biotin/streptavidin system.
Authors: Liu, Xuewu and Li, Xingfei and Xie, Zhengjun and Zhou, Xing and Chen, Long and Qiu, Chao and Lu, Cheng and Jin, Zhengyu and Long, Jie
Journal: International journal of biological macromolecules (2024): 129807
Reports on the sensitivity enhancement in interferometric based biosensors by biotin-streptavidin system.
Authors: Murillo, A M M and Holgado, M and Laguna, M
Journal: Heliyon (2023): e23123
An ultrasensitive aptasensor for exosomes detection based on biotin-streptavidin and MXenes.
Authors: Wang, Zhangmin and Zhang, Ze and Pan, Hongzhi and Chang, Dong
Journal: Analytical biochemistry (2023): 115233
An electrochemiluminescence aptasensor based on highly luminescent silver-based MOF and biotin-streptavidin system for mercury ion detection.
Authors: Liu, Si-Qi and Chen, Jing-Shuai and Liu, Xing-Pei and Mao, Chang-Jie and Jin, Bao-Kang
Journal: The Analyst (2023): 772-779
Manganese Transfer Hydrogenases Based on the Biotin-Streptavidin Technology.
Authors: Wang, Weijin and Tachibana, Ryo and Zou, Zhi and Chen, Dongping and Zhang, Xiang and Lau, Kelvin and Pojer, Florence and Ward, Thomas R and Hu, Xile
Journal: Angewandte Chemie (International ed. in English) (2023): e202311896
Impact of Biotin Supplementation on Human Chorionic Gonadotropin Immunoassays Utilizing Biotin-Streptavidin Binding Methods in Urine.
Authors: Goodrum, Jenna M and Nair, Vinod S and Moore, Chad and Crouch, Andre K and Eichner, Daniel and Miller, Geoffrey D
Journal: Clinical chemistry (2023): 754-762
Development of a Novel Recombinant Full-Length IgY Monoclonal Antibody against Human Thymidine Kinase 1 for Automatic Chemiluminescence Analysis on a Sandwich Biotin-Streptavidin Platform for Early Tumour Discovery.
Authors: Wang, Xiangbin and Li, Shan and Zhao, Rui and Li, Huijun and Gao, Peng and Jin, Cuicui and Fang, Cong and Liu, Yongping and Hei, Ailian and Zhou, Ji and Li, Jin and He, Ellen and Skog, Sven
Journal: Journal of immunology research (2023): 7612566
Triple quantitative detection of three inflammatory biomarkers with a biotin-streptavidin-phycoerythrin based lateral flow immunoassay.
Authors: Wang, Xiao-Ming and Li, Shan and Li, Lin-Hai and Song, Jian-Xun and Lu, Yan-Hua and Zhou, Zhi-Wei and Zhang, Lei
Journal: Analytical biochemistry (2022): 114915