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

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<p>The mechanism of Buccutite™ bioconjugation system used for ReadiLink™ Peroxidase Antibody Conjugation Kit (Cat# 5504).</p>
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Unit Size: Cat No: Price (USD): Qty:
1 kit 5504 $750

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Storage Refrigerated (2-4 °C)
Category Protein Biochemistry
General proteins
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.


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This protocol only provides a guideline, and should be modified according to your specific needs.

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) and Spin Column (Component D). 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.


1. Prepare antibody solution :

For labeling 100 µg antibody (assuming the target antibodyconcentration is 1 mg/mL), mix 5 µL (5% of the total reaction volume) of Reaction Buffer (Component C) with 100 µL of the target antibody solution.

Note 1. If you have a difference  concentration, adjust the antibody volume accordingly to make ~100 µg antibody available for your labeling reaction.

Note 2: 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 Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa (cat# UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation.

Note 3: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.

Note 4: The antibody –Buccutite™ MTA reaction efficiency is significantly reduced if the antibody concentration is less than 1 mg/mL. For optimal labeling efficiency the final antibody concentration range of 1-10 mg/mL is recommended.

2. Run Antibody-Buccutite™ MTA reaction:

2.1    Add the antibody solution directly into the vial of Buccutite ™ MTA (Component B), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

2.2    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.

3. Prepare spin column for antibody-Buccutite™ MTA purification:

3.1    Invert the provided spin column (Component D) several times to re-suspend the settled gel and remove any bubbles.

3.2    Snap off the tip and place the column in a washing tube (2 mL, not provided). 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).

3.3    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.

3.4    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.

3.5    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.

4. Purify the antibody-Buccutite™ MTA solution:

4.1    Place the column (from Step 3.5) in a clean Collecting Tube (1.5 mL, not provided). Carefully load the sample (~105 μL, from Step 2.2) directly to the center of the column.

4.2    After loading the sample, add 5 μL of 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 antibody-Buccutite™ MTA solution.

5. Make HRP-antibody conjugation:

5.1    Make HRP- Buccutite™ FOL solution by adding 50 μL ddH2O into the vial of HRP- Buccutite™ FOL (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

5.2    Mix whole vial of HRP- Buccutite™ FOL solution (from Step 5.1) into the purified antibody- Buccutite™ MTA solution (from Step 4.2), mix well and rotating the mixture for 1 hour at room temperature.

5.3    The HRP-antibody conjugate is now ready to use.

Note 1: For immediate use, the HRP-antibody conjugate need be diluted with the buffer of your choice.

Note 2: For longer term storage, HRP-antibody conjugate solution need be concentrated or freeze dried.

References & Citations

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

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

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

Additional Documents

Safety Data Sheet (SDS)

1. Fluorescent Labeling Probes & Kits

Application Notes
1. AssayWise Letters 2014, Vol 3(2)

Certificate of Analysis