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Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit *Optimized for Labeling 1 mg Protein*

The mechanism of Buccutite™ bioconjugation system used for ReadiLink™ Peroxidase Antibody Conjugation Kit (Cat# 5504).
The mechanism of Buccutite™ bioconjugation system used for ReadiLink™ Peroxidase Antibody Conjugation Kit (Cat# 5504).
Ordering information
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Catalog Number5504
Unit Size
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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


Protein-protein conjugations are commonly performed with a bifunctional linker (such as the commonly used SMCC), having different reactivity on each end for linking two different proteins. One end of the crosslinker reacts (via NHS ester) with amines (-NH2) found in the amino acid lysine and N-terminus, and the other end reacts (via maleimide) with the thiol groups (-SH) found in the amino acid cysteine. However, SMCC-modified protein is extremely unstable and often self-reactive since proteins often contain both amine and thiol groups that cause significant amount of homo-crosslinking. In addition it is quite difficult and tedious to quantify the number of maleimide groups on a protein. Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit is designed for preparing horseradish peroxidase (HRP) conjugates directly from proteins, peptides, and other ligands that contain a free amino group. The HRP provided in our kit has been pre-activated with our proprietary linker Buccutite™ FOL, and can be directly used for conjugation. The entire process only requires two simple mixings without further purification required. The Buccutite™ FOL-activated HRP readily reacts with Buccutite™ MTA-containing molecules under extremely mild neutral conditions without any catalyst required. Compared to commonly used SMCC and other similar technologies, our Buccutite™ bioconjugation system is much more robust and easier to use. It enables faster and quantitative conjugation of biomolecules with higher efficiencies and yields.


Component A: Buccutite™ FOL-Activated HRP1 vial (lyophilized)
Component B: Buccutite™ MTA1 vial (lyophilized)
Component C: Reaction Buffer1 vial (200 µL)

Example protocol


Protocol Summary
  1. Add 10 µL Reaction Buffer (Component C) into antibody solution (200 µL) if antibody concentration is 5 mg/mL in PBS
  2. Add 10 µL DMSO to Buccutite™ MTA vial (Component B) to make Buccutite™ MTA working solution
  3. Add 4.5 µL Buccutite™ MTA stock solution into the antibody solution
  4. Incubate at room temperature for 30 minutes
  5. Add 500 µL H2O to reconstitute Buccutite™ FOL-Activated HRP (Component A)
  6. Mix the Buccutite™ MTA activated antibody solution with 500 µL Buccutite™ FOL-Activated HRP (Component A)
  7. Incubate at room temperature for 60 minutes 
Important      Upon receipt, store the kit at 4 oC. When stored properly, the kit should be stable for six months. Alternatively Components A and B can be stored at -20 °C. Do not freeze Reaction Buffer (Component C). Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following SOP is an example for labeling goat anti-mouse IgG antibody.


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.

Buccutite™ MTA stock solution
Add 10 µL of DMSO into the vial of Buccutite™ MTA (Component B).
Note      This stock solution should be used promptly.


Antibody working solution
For labeling 1 mg antibody (assuming the target antibody concentration is 5 mg/mL), mix 10 µL of Reaction Buffer (Component C) with 200 µL of the target antibody solution. If your antibody is not 5 mg/ml, please add 5% of total volume of Reaction Buffer (Component C).
Note      The protocol assuming the target antibody concentration is 5 mg/mL. The antibody –Buccutite™ MTA reaction efficiency is significantly reduced if the antibody concentration is less than 1 mg/mL. For optimal labeling efficiency the antibody concentration range of 1-5 mg/mL is recommended.
Note      The antibody should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4; If the antibody is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, or use ReadiUse™ 10KD Spin Filter (Cat. # 60502 from AAT Bioquest) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation. 
Note      Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.


Run Antibody-Buccutite™ MTA reaction
  1. Add 4.5 µL Buccutite ™ MTA stock solution to antibody working solution, and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
  2. Keep the antibody- Buccutite ™ MTA reaction mixture at room temperature for 30 - 60 minutes.
    Note      The antibody-Buccutite™ MTA reaction mixture can be rotated or shaken for longer time if desired. 

Make HRP-antibody conjugation
  1. Make HRP- Buccutite™ FOL solution by adding 500 µL ddH2O into the vial of Buccutite™ FOL-Activated HRP (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.
  2. Mix whole vial of Buccutite™ FOL-Activated HRP solution into the antibody- Buccutite™ MTA solution, mix well and rotating the mixture for 1 hour at room temperature.
  3. The HRP-antibody conjugate is now ready to use. Optional: HRP-antibody conjugate can be further purified through size exclusion chromatography to get better performance.
    Note     Alternatively, add antibody- Buccutite™ MTA solution mixture to the vial of Buccutite™ FOL-Activated HRP directly. 

Storage of HRP-Antibody 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 HRP-Antibody conjugate solution could be stored at 4 °C for two months and kept from light. For longer storage, the HRP-antibody conjugates could be lyophilized and stored at ≤ –20 °C.


View all 9 references: Citation Explorer
Influence of the antibody-peroxidase coupling methods on the conjugate stability and on the methodologies for the preservation of the activity in time
Authors: Presentini R, Terrana B.
Journal: J Immunoassay (1995): 309
Comparative activity of peroxidase-antibody conjugates with periodate and glutaraldehyde coupling according to an enzyme immunoassay
Authors: Tresca JP, Ricoux R, Pontet M, Engler R.
Journal: Ann Biol Clin (Paris) (1995): 227
Peroxidase labelled monoclonal antibody against light chains of human cardiac myosin
Authors: Strakova Z, Barancik M, Lukacova D, Angyal R, Slosarcikova L, Horakova K.
Journal: Gen Physiol Biophys (1991): 63
Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays
Authors: Tijssen P, Kurstak E.
Journal: Anal Biochem (1984): 451
Direct immunocytochemistry with a horseradish peroxidase-conjugated monoclonal antibody against substance P
Authors: Boorsma DM, Cuello AC, van Leeuwen FW.
Journal: J Histochem Cytochem (1982): 1211
Comparison between peroxidase-conjugated antigen or antibody and peroxidase-anti-peroxidase complex in a postembedding procedure
Authors: Tougard C, Tixier-Vidal A, Avrameas S.
Journal: J Histochem Cytochem (1979): 1630
The unlabeled antibody enzyme method. Attempted use of peroxidase-conjugated antigen as the third layer in the technique
Authors: Sternberger LA, Petrali JP.
Journal: J Histochem Cytochem (1977): 1036
Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate
Authors: Broorsma DM, Steefkerk JG, Kors N.
Journal: J Histochem Cytochem (1976): 1017
Peroxidase-labeled antibody. A new method of conjugation
Authors: Nakane PK, Kawaoi A.
Journal: J Histochem Cytochem (1974): 1084