logo
AAT Bioquest

ReadiLink™ Protein Biotinylation Kit *Powered by ReadiView™ Biotin Visionization Technology*

ReadiView™ biotin conjugated with specially designed color tag (CT) for easy determination of biotinylation degree. 
ReadiView™ biotin conjugated with specially designed color tag (CT) for easy determination of biotinylation degree. 
ReadiView™ biotin conjugated with specially designed color tag (CT) for easy determination of biotinylation degree. 
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
QuotationRequest
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
Related products
ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid mFluor™ Violet 420 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid mFluor™ Violet 510 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid mFluor™ Violet 540 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid mFluor™ Blue 570 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid mFluor™ Green 620 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid mFluor™ Yellow 630 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid mFluor™ Red 700 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid mFluor™ Red 780 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 350 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 555 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 594 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 680 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 700 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 750 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 633 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid iFluor® 790 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid FITC Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid trFluor™ Eu Antibody Labeling Kit *Microscale Optimized for Labeling 50 ug Antibody Per Reaction*
ReadiLink™ Rapid trFluor™ Tb Antibody Labeling Kit *Microscale Optimized for Labeling 50 ug Antibody Per Reaction*
ReadiLink™ Protein Conjugation Stop Buffer
ReadiLink™ BSA Conjugation Kit
ReadiLink™ KLH Conjugation Kit
ReadiLink™ Rapid Cy3 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid Cy5 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ Rapid Cy7 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*
ReadiLink™ xtra Rapid iFluor® 350 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid iFluor® 488 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid iFluor® 555 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid iFluor® 594 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid iFluor® 647 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid iFluor® 750 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid FITC Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid Cy3 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid Cy5 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid Cy7 Antibody Labeling Kit *BSA-Compatible*
ReadiLink™ xtra Rapid XFD594 Antibody Labeling Kit *BSA-Compatible, XFD594 Same Structure to Alexa Fluor™ 594*
ReadiLink™ Rapid XFD488 Antibody Labeling Kit *XFD488 Same Structure to Alexa Fluor™ 488*
ReadiLink™ Rapid XFD555 Antibody Labeling Kit *XFD555 Same Structure to Alexa Fluor™ 555*
ReadiLink™ Rapid XFD594 Antibody Labeling Kit *XFD594 Same Structure to Alexa Fluor™ 594*
ReadiLink™ Rapid XFD647 Antibody Labeling Kit *XFD647 Same Structure to Alexa Fluor™ 647*
ReadiLink™ Rapid XFD750 Antibody Labeling Kit *XFD750 Same Structure to Alexa Fluor™ 750*
ReadiLink™ iFluor® 488 Nick Translation dsDNA Labeling Kit
ReadiLink™ iFluor® 555 Nick Translation dsDNA Labeling Kit
ReadiLink™ iFluor® 647 Nick Translation dsDNA Labeling Kit
ReadiLink™ Cy3 Nick Translation dsDNA Labeling Kit
ReadiLink™ Cy5 Nick Translation dsDNA Labeling Kit
ReadiLink™ Biotin Nick Translation dsDNA Labeling Kit
ReadiLink™ DIG (Digoxigenin) Nick Translation dsDNA Labeling Kit
ReadiLink™ iFluor® 488 Oligo and ssDNA Labeling Kit
ReadiLink™ iFluor® 555 Oligo and ssDNA Labeling Kit
ReadiLink™ iFluor® 647 Oligo and ssDNA Labeling Kit
ReadiLink™ Cy3 Oligo and ssDNA Labeling Kit
ReadiLink™ Cy5 Oligo and ssDNA Labeling Kit
ReadiLink™ Biotin Oligo and ssDNA Labeling Kit
ReadiLink™ DIG (Digoxigenin) Oligo and ssDNA Labeling Kit
ReadiLink™ xtra Rapid XFD488 Antibody Labeling Kit *BSA-Compatible, XFD488 Same Structure to Alexa Fluor™ 488*
ReadiLink™ xtra Rapid XFD555 Antibody Labeling Kit *BSA-Compatible, XFD555 Same Structure to Alexa Fluor™ 555*
ReadiLink™ xtra Rapid XFD647 Antibody Labeling Kit *BSA-Compatible, XFD647 Same Structure to Alexa Fluor™ 647*
ReadiLink™ xtra Rapid XFD750 Antibody Labeling Kit *BSA-Compatible, XFD750 Same Structure to Alexa Fluor™ 750*
ReadiLink™ iFluor® 488 FISH Fluorescence Imaging Kit
ReadiLink™ iFluor® 555 FISH Fluorescence Imaging Kit
ReadiLink™ iFluor® 647 FISH Fluorescence Imaging Kit
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 945633-30-7*
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
Amplite® Colorimetric Biotin Quantitation Kit
Biotin-11-dUTP *1 mM in Tris Buffer (pH 7.5)* *CAS 86303-25-5*
Biotin-16-dUTP *1 mM in Tris Buffer (pH 7.5)* *CAS 136632-31-0*
Biotin-20-dUTP *1 mM in Tris 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
Biotin-11-dGTP *1 mM in Tris Buffer (pH 7.5)*
Biotin NTA
Biotin-X IDA
ReadiCleave™ SSL biotin NHS ester
Biotin-dT Phosphoramidite
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] *10 mM*
Biotin PEG4 amine
Show More (113)

OverviewpdfSDSpdfProtocol


Biotin is widely used for labeling biomolecules, in particular, antibodies. This kit is primarily used for the preparation of biotin-labeled IgG for enzyme immunoassay (EIA). Our kit uses our ReadiView™ biotin succinimidyl ester (#3059) that reacts with an amino group of IgG and other biomolecules. Our unique biotin contained in the kit carries a color tag for indicating the degree of biotinylation, thus eliminating the troublesome HABA biotinylation determination. This kit contains all of the necessary reagents for labeling and purification. On our hands, 5 to 8 biotin molecules can be conjugated to each IgG molecule using our kit.

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Add 5 µL Reaction Buffer (Component B) into target protein (50 µL)
  2. Add the protein solution into ReadiView™ Biotin SE (Component A)
  3. Incubate at room temperature for 30-60 minutes
  4. Purify the conjugate by spin column 
Important      Upon receipt, store ReadiView™ Biotin SE (Component A) at -20 °C, kept from moisture. Store other components at room temperature. Do not freeze Reaction Buffer (Component B) and Spin Column (Component D). Warm all the components before opening, and immediately prepare the required solutions before starting the conjugation. You might need further optimization for your protein labeling since this SOP was developed for Goat anti-Rabbit IgG labeling.

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.

ReadiView™ Biotin SE stock solution
Add 10 µL of DMSO (Component C) into the vial of ReadiView™ Biotin SE (Component A), and vortex them vigorously.
Note     Prepare the ReadiView™ Biotin SE stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the ReadiView™ Biotin SE stock solution may reduce the biotin activity. Solution B can be stored in freezer for two weeks when kept from moisture.
Note     Aliquot the ReadiView™ Biotin SE stock solution into 5 vials (2 µL/vial). ONLY one vial is needed for labeling 100 µg proteins. The remaining vials can be stored in freezer for 2 weeks in case you need to repeat your conjugation.

PREPARATION OF WORKING SOLUTION

Protein working solution
For labeling 100 µg target protein (assuming the target protein concentration is 2 mg/mL), mix 5 µL (10% of the total reaction volume) of Reaction Buffer (Component B) with 50 µL of the target protein solution.
Note     If you have a different protein concentration, adjust the protein volume accordingly to make ~2 mg protein available for this labeling reaction.
Note     The pH of the protein solution 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 Reaction Buffer (Component B) or saturated sodium bicarbonate solution.
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 might 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.

SAMPLE EXPERIMENTAL PROTOCOL

Run conjugation reaction
  1. Add the protein working solution into the vial of ReadiView™ Biotin SE stock solution (2 µL/vial), and mix them well by repeatedly pipetting for a few times or vortex the vial for 2-5 minutes.
  2. Keep the conjugation reaction mixture at room temperature for 30 - 60 minutes.
    Note      The conjugation reaction mixture can be rotated or shaken for longer time if desired. 

Prepare spin column for sample purification
  1. Invert the provided spin column (Component D) several times to re-suspend the settled gel and remove any bubbles.
  2. Snap off the tip and place the column in a washing tube (2 mL, Component E). Remove the cap to allow the excess packing buffer to drain by gravity to the top of the gel bed. If column does not begin to flow, push cap back into column and remove it again to start the flow. Discard the drained buffer, and then place the column back into the Washing Tube. However, centrifuge immediately if the column is placed into a 12 x 75 mm test tube (not provided).
  3. Centrifuge for 1 minutes in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer.
  4. Apply 1-2 mL 1X PBS (pH 7.2-7.4) to the column. After each application of PBS, let the buffer drain out by gravity, or centrifuge the column for 2 minutes to remove the buffer. Discard the buffer from the collection tube. Repeat this process for 3-4 times.
  5. Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer. 

Purify the conjugates
  1. Place the column (from Step Prepare spin column for sample purification) in a clean Collecting Tube (1.5 mL, Component F). Carefully load the sample (20-100 µL, from Step conjugation reaction) directly to the center of the column.
  2. After loading the sample, add 1X PBS (pH 7.2-7.4) to make the total volume of 110 µL. Centrifuge the column for 5-6 minutes at 1,000 x g, and collect the solution that contains the desired biotin-labelled protein. 

Storage of Protein Conjugate
The antibody conjugate should be stored at > 0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin). The conjugate solution could be stored at 4 °C for two months without significant change when stored in the presence of 2 mM sodium azide and kept from light. For longer storage, the protein conjugates could be lyophilized or divided into single-used aliquotes and stored at ≤ –60 °C.

Centrifugation Notes
Spin column (Component D) can fit into 2 mL microcentrifuge tubes or 12 x 75 mm test tubes for sample collection during centrifugation. Use the 2 mL microtube with the columns for the initial column equilibration step.
Swinging bucket centrifuges capable of generating a minimum force of 1,000 x g are suitable for Bio-Spin column use. The gravitational force created at a particular revolution speed is a function of the radius of the microcentrifuge rotor. Consult the swinging bucket centrifuge instruction manual for the information about conversion from revolutions per minute (RPM) to centrifugal or g-force. Alternatively, use the following equation to calculate the speed in RPM required to reach the gravitational force of 1,000 x g.
RCF (x g) = (1.12 x 10-5)×(RPM)×2×r (RCF is the relative centrifugal force, r is the radius in centimeters measured from the center of the rotor to the middle of the Bio-Spin column, and RPM is the speed of the rotor).

Degree of Biotinylation
The degree of biotinylation can be readily calculated by the following equation with a simple absorption spectrum:
Number of Biotin/Conjugate = [A360/9900] ÷ [A280protein]
A360 and A280 are the absorbances of the conjugation at 360 and 280 nm respectively, and εprotein is the extinction coefficient of the antibody or protein to be labeled.

Images


References


View all 62 references: Citation Explorer
Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein
Authors: Volkmann G, Liu XQ.
Journal: PLoS One (2009): e8381
Tandem affinity purification of protein complexes in mouse embryonic stem cells using in vivo biotinylation
Authors: Wang J, Cantor AB, Orkin SH.
Journal: Curr Protoc Stem Cell Biol (2009): Unit1B 5
A multifunctional single-attachment-point reagent for controlled protein biotinylation
Authors: Garanger E, Weissleder R, Josephson L.
Journal: Bioconjug Chem (2009): 170
Measuring plasma membrane protein endocytic rates by reversible biotinylation
Authors: Gabriel L, Stevens Z, Melikian H.
Journal: J Vis Exp. (2009)
Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells
Authors: Kim J, Cantor AB, Orkin SH, Wang J.
Journal: Nat Protoc (2009): 506
Protein-protein interaction detection in vitro and in cells by proximity biotinylation
Authors: Fern, undefined and ez-Suarez M, Chen TS, Ting AY.
Journal: J Am Chem Soc (2008): 9251
Analysis of apolipoprotein E nuclear localization using green fluorescent protein and biotinylation approaches
Authors: Kim WS, Elliott DA, Kockx M, Kritharides L, Rye KA, Jans DA, Garner B.
Journal: Biochem J (2008): 701
Use of protein biotinylation in vivo for immunoelectron microscopic localization of a specific protein isoform
Authors: Viens A, Harper F, Pichard E, Comisso M, Pierron G, Ogryzko V.
Journal: J Histochem Cytochem (2008): 911
Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives
Authors: Vila A, Tallman KA, Jacobs AT, Liebler DC, Porter NA, Marnett LJ.
Journal: Chem Res Toxicol (2008): 432
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 4: DNA Biotinylation, Precipitation, and Arraying of Samples
Authors: Link AJ, Labaer J.
Journal: CSH Protoc (2008): pdb prot5059