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ReadiView™ biotin succinimidyl ester
Biotin/avidin complexes are widely applied for a variety of biological detections. Although a large number of biotin-labeled bioconjugates are commercially available, the accurate determination of biotinylation degree (ratio of biotin/biopolymer) is still a great challenge for biochemists. HABA is still predominantly used for determining the degree of biotinylation (through its absorption with the extinction coefficient = 34,000/M-1cm-1). When a biotin-containing sample is added, the biotin binds strongly to avidin and displaces the weakly bound HABA. The resulting decrease in absorbance relates to the amount of biotin. However there are many factors that affect the accuracy of HABA method, making this method unreliable for many biotin-labeled conjugates. Our ReadiView™ biotin contains specially designed Color Tag (CT) that makes the biotinylation degree readily accessible by simply calculating the corrected absorption ratio of 280 nm/385 nm. Our specially designed tag has very minimal effect on the biotin binding affinity, and its absorption maximum was designed to make the tag have minimal quenching effect on the most fluorophores that are used for labeling avidins.
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.
1. Protein stock solution (Solution A)
Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution. Note: The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer. Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results. Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.2. ReadiView™ biotin succinimidyl ester stock solution (Solution B)
Add anhydrous DMSO into the vial of ReadiView™ biotin succinimidyl ester to make a 10 mM stock solution. Mix well by pipetting or vortex. Note: Prepare the ReadiView™ biotin succinimidyl ester (Solution B) before starting the conjugation. Use promptly. Extended storage of the ReadiView™ biotin succinimidyl ester stock solution may reduce its activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.SAMPLE EXPERIMENTAL PROTOCOL
This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with ReadiView™ biotin succinimidyl ester. You might need further optimization for your particular proteins. Note: Each protein requires distinct ReadiView™ biotin succinimidyl ester/protein ratio, which also depends on the properties of ReadiView™ biotin succinimidyl ester. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low ReadiView™ biotin succinimidyl ester/protein ratio gives reduced sensitivity.
Run conjugation reaction
- Use 10:1 molar ratio of Solution B (ReadiView™ biotin succinimidyl ester)/Solution A (protein) as the starting point: Add 5 µL of the ReadiView™ biotin succinimidyl ester stock solution (Solution B, assuming the ReadiView™ biotin succinimidyl ester stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD. Note: We recommend to use 10:1 molar ratio of Solution B (ReadiView™ biotin succinimidyl ester)/Solution A (protein). If it is too less or too high, determine the optimal ReadiView™ biotin succinimidyl ester/protein ratio at 5:1, 15:1 and 20:1 respectively.
- Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.
Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.- Prepare Sephadex G-25 column according to the manufacture instruction.
- Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
- Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
- Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired ReadiView™ biotin-protein conjugate. Note: For immediate use, the ReadiView™ biotin-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses. Note: For longer term storage, ReadiView™ biotin-protein conjugate solution need be concentrated or freeze dried.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of ReadiView™ 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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 142.084 µL | 710.419 µL | 1.421 mL | 7.104 mL | 14.208 mL |
| 5 mM | 28.417 µL | 142.084 µL | 284.168 µL | 1.421 mL | 2.842 mL |
| 10 mM | 14.208 µL | 71.042 µL | 142.084 µL | 710.419 µL | 1.421 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Citations
View all 3 citations: Citation Explorer
A low-cost, high-performance system for fluorescence lateral flow assays
Authors: Lee, Linda G and Nordman, Eric S and Johnson, Martin D and Oldham, Mark F
Journal: Biosensors (2013): 360--373
Authors: Lee, Linda G and Nordman, Eric S and Johnson, Martin D and Oldham, Mark F
Journal: Biosensors (2013): 360--373
Implementation of P22 viral capsids as nanoplatforms
Authors: Kang, Sebyung and Uchida, Masaki and O'Neil, Alison and Li, Rui and Prevelige, Peter E and Douglas, Trevor
Journal: Biomacromolecules (2010): 2804--2809
Authors: Kang, Sebyung and Uchida, Masaki and O'Neil, Alison and Li, Rui and Prevelige, Peter E and Douglas, Trevor
Journal: Biomacromolecules (2010): 2804--2809
Calcium-dependent activation of transglutaminase 2 by nanosecond pulsed electric fields
Authors: Morotomi-Yano, Keiko and Yano, Ken-ichi
Journal: FEBS Open Bio
Authors: Morotomi-Yano, Keiko and Yano, Ken-ichi
Journal: FEBS Open Bio
References
View all 78 references: Citation Explorer
Evaluation of a new biotin-DOTA conjugate for pretargeted antibody-guided radioimmunotherapy (PAGRIT((R)))
Authors: Urbano N, Papi S, Ginanneschi M, De Santis R, Pace S, Lindstedt R, Ferrari L, Choi S, Paganelli G, Chinol M.
Journal: Eur J Nucl Med Mol Imaging. (2006)
Authors: Urbano N, Papi S, Ginanneschi M, De Santis R, Pace S, Lindstedt R, Ferrari L, Choi S, Paganelli G, Chinol M.
Journal: Eur J Nucl Med Mol Imaging. (2006)
A biotin-protein bond with stability in plasma
Authors: Bogusiewicz A, Mock NI, Mock DM.
Journal: Anal Biochem (2005): 98
Authors: Bogusiewicz A, Mock NI, Mock DM.
Journal: Anal Biochem (2005): 98
Instability of the biotin-protein bond in human plasma
Authors: Bogusiewicz A, Mock NI, Mock DM.
Journal: Anal Biochem (2004): 156
Authors: Bogusiewicz A, Mock NI, Mock DM.
Journal: Anal Biochem (2004): 156
Design, synthesis, and evaluation of 5'-S-aminoethyl-N(6)- azidobenzyl-5'-thioadenosine biotin conjugate: a bifunctional photoaffinity probe for the es nucleoside transporter
Authors: Addo JK, Buolamwini JK.
Journal: Bioconjug Chem (2004): 536
Authors: Addo JK, Buolamwini JK.
Journal: Bioconjug Chem (2004): 536
Biotin is endogenously expressed in select regions of the rat central nervous system
Authors: McKay BE, Molineux ML, Turner RW.
Journal: J Comp Neurol (2004): 86
Authors: McKay BE, Molineux ML, Turner RW.
Journal: J Comp Neurol (2004): 86
Page updated on December 17, 2024
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