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Protein to Protein Conjugation

Buccutite™ crosslinking technology offers a truly unique and trouble-free method of labeling antibodies with another macromolecule, such as an enzyme or phycobiliprotein. Compared to conventional techniques for protein-protein conjugation, such as the labor intensive SMCC chemical labeling method, Buccutite™ conjugation is significantly more robust and less demanding. These kits allow optimal labeling of microgram or milligram amounts of antibodies in less than two hours, with no purification necessary and 100% recovery of antibody. With Buccutite™ labeling kits, routinely prepare covalently-labeled conjugates that can tolerate the rigorous handling and washing that is required in a typical immunoassay (e.g. ELISA, IHC and Western blot) or flow cytometry application. AAT Bioquest offers Buccutite™ antibody labeling kits for preparing horseradish peroxidase (HRP), alkaline phosphatase (AP), PE, APC, PerCP and tandem dye antibody conjugates.

Too busy to perform your own conjugation or having difficulty finding the right labeling kit? Not to worry AAT Bioquest's bioconjugation service can prepare the custom antibody conjugate you need.

 

 

Buccutite™ vs SMCC Conjugation At-A-Glance


Conjugation Method Buccutite™ Kits SMCC Conjugation
Minimum antibody concentration 100 µg/mL 0.5 to 5 mg/mL
Labeling Chemistry Uses two propietary linkers, Buccutite™ MTA and Buccutite™ FOL, to facilitate protein-protein conjugation. Uses a heterobifunctional crosslinker to facilitate protein-protein conjugation. Labeling protocol is tedioius and requries existing knowledge of bioconjugation chemistry.
Available labels PE, APC, Tandem Dyes, HRP, Poly-HRP and AP. Cat No 1315 can be used to conjugate any two proteins. User is responsible for supplying proteins, method suitable for any protein-protein conjugation
Type of bond between label and antibody Covalent Covalent
Compatible with BSA or other stabilizers? No No
Requires purification? No Yes
Time required for conjugation 2 hrs 4 hrs or more
Hands-on-time ~15 minutes 2 hrs
Conjugate yield 100 % ~30%
Batch-to-batch variation Minimal High
Applications¹ FC, IF, IHC, WB, ELISA FC, IF, IHC, WB, ELISA
  1. FC = flow cytometry; IF = immunofluorescence; IHC = immunohistochemistry; WB = Western blot; ELISA = enzyme-linked immunosorbent assay.

 

Buccutite™ HRP and Poly-HRP Antibody Labeling Kits



Buccutite™ Peroxidase (HRP) Antibody Labeling Kit workflow (figure drawn in BioRender).
Buccutite™ Peroxidase (HRP) Antibody Labeling Kits enable researchers to efficiently label primary antibodies with the detection enzyme horseradish peroxidase (HRP) in less than 2 hours. To streamline the process, the HRP provided in these kits are preactivated with our proprietary linker Buccutite™ FOL, and can be directly used for antibody conjugation. Simply activate the antibody or protein you wish to label with the Buccutite™ MTA linker provided in each kit, and mix together with the Buccutite™ FOL-activated HRP. The resulting enzyme labeled antibody conjugates are ideal for various immunoassay applications including ELISA, IHC and western blotting, and are highly stable for long-term storage. Buccutite™ Peroxidase Antibody Labeling Kits are available in three sizes optimized for labeling 25 µg, 100 µg and 1 mg of antibody per labeling reaction.

For difficult applications that require an additional level of sensitivity, we offer Buccutite™ Poly-HRP Antibody Labeling kits. These kits enable the direct labeling of antibodies to polymers of HRP, resulting in conjugates that deliver the highest sensitivity and signal-to-noise ratios. Poly-HRP conjugates are ideal for applications where the target of interest is in low-abundance or when sample volume is limited.

Key features of the Buccutite™ HRP and Poly-HRP Antibody Labeling Kits

  • Can effectively and efficiently label antibody concentrations as low as 100 µg/mL (SMCC requires at least 1 mg/mL)
  • Labeled antibodies are ready in under 2 hours, with as little as ~15 minutes hands on-time
  • Simple two step mixing protocol reduces batch-to-batch variation
  • No column purification step required
  • 100% conjugation yield
  • Conjugates stable for long-term storage (4 °C, 12 months)
  • Conjugates can be used for ELISA, IHC, blotting, Power Styramide Signal Amplification and TSA Imaging
  • Offers flexibility in experimental design, HRP conjugates are compatible with colorimetric, fluorimetric and chemiluminescent substrates

High-performance alternative to Lightning-Link™ HRP Labeling Kit



Direct ELISA curves were generated using the HRP-goat-anti-mouse IgG conjugate prepared using the Buccutite™ Peroxidase Antibody Labeling Kit and Lightning-Link® HRP Antibody Labeling Kit. Mouse monoclonal antibody (0-10 ng/well) was coated on a 96-well plate overnight, and then blocked with PBST and 2% BSA. Wells were washed and then incubated with 100 µL/well GAM IgG-HRP conjugate (100 ng/mL) for one hour. Wells were were washed again and then a TMB substrate solution (Cat No. 11012) was used to detect the immobilized mouse IgG with 30 minute incubation and read at 405 nm.
 

Table 1. Buccutite™ Peroxidase Antibody Labeling kits designed for labeling antibodies with HRP and poly-HRP.

Name
Labeling Size / Reaction
Size
Cat No.
Buccutite™ Peroxidase (HRP) Antibody Conjugation KitOptimized for Labeling 25 µg Protein2 Labelings5505
Buccutite™ Peroxidase (HRP) Antibody Conjugation KitOptimized for Labeling 100 µg Protein2 Labelings5503
Buccutite™ Peroxidase (HRP) Antibody Conjugation KitOptimized for Labeling 1 mg Protein1 Labeling5504
Buccutite™ Peroxidase (HRP) Antibody Conjugation KitOptimized for Labeling 1 mg Protein5 Labeling5504
Buccutite™ Poly-HRP Antibody Conjugation KitOptimized for Labeling 50 µg Protein1 Labeling5518
Buccutite™ Poly-HRP Antibody Conjugation KitOptimized for Labeling 50 µg Protein2 Labelings5519

 

Buccutite™ Rapid PE, APC, PerCP and Tandem Dye Antibody Labeling Kits



Buccutite™ Rapid Antibody Labeling Kit workflow (figure drawn in BioRender).
Phycobiliproteins, such as phycoerythrin (PE), allophycocyanin (APC) and peridinin-chlorophyll-protein (PerCP), have the capacity to generate fluorescence signals significantly brighter than any commercially available synthetic fluorophore. When conjugated to molecules having biological specificity, such as antibodies, protein A or streptavidin, phycobiliproteins yield extraordinarily luminescent probes, that have found considerable success in fluorescence imgaging applications that require high sensitivity but not photostability, primarily flow cytometry, FACS and immunophenotyping. Moreover, phycobiliproteins can be labeled with small organic fluorophores to generate tandem dyes with exceptionally long Stoke's Shifts for multiparametric analysis using a single excitation source. For example, FITC, PE, PE-Cy5 and PE-Cy7 are ideal for multiparametric analysis since all four fluorophores can simultaneously be excited using the 488 nm laser line and they all demonstrate excellent spectral separation.

Buccutite™ Rapid Antibody Labeling Kits makes preparing antibodies for flow cytometry quick and easy. Using a simple two step mixing protocol, researchers can directly conjugate PE, APC, PerCP or a tandem dye to any antibody or protein in less than 2 hours. No column purification is necessary, so you recovery is always 100%. Buccutite™ Rapid Antibody Labeling Kits are available in three sizes optimized for labeling 25 µg, 100 µg or 1 mg of antibody per labeling reaction.

Key features of the Buccutite™ Rapid Antibody Labeling Kits

  • Can effectively and efficiently label antibody concentrations as low as 100 µg/mL (SMCC requires at least 1 mg/mL)
  • Labeled antibodies are ready in under 2 hours, with as little as ~15 minutes hands on-time
  • Simple two step mixing protocol reduces batch-to-batch variation
  • No column purification step required
  • 100% conjugation yield
  • Conjugates stable for long-term storage (4 °C, 12 months)
  • Conjugtes ideal for multicolor flow cytometry applications

High-performance alternative to Lightning-Link™ Labeling Kits



Flow cytometry analysis of human PBMC cells. Anti-human CD4 antibody prepared with Buccutite™ Rapid PE Antibody Labeling Kit (Cat No. 1310) or Lightning-Link® PE. The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the FITC channel.
Labeling Kit CD4(SK3)- Conjugate Stain Index
Buccutite™ Kit CD4-PE Conjugate 55
Lightning-Link® Kit CD4-PE Conjugate 28
 

Table 2. Buccutite™ Antibody Labeling kits designed for labeling antibodies with fluorescent proteins and tandem dyes.

Name
Labeling Size / Reaction
Ex (nm)
Em (nm)
Size
Cat No.
Buccutite™ Rapid PE Antibody Labeling KitOptimized for Labeling 25 µg Protein5655752 Labelings1312
Buccutite™ Rapid PE Antibody Labeling KitOptimized for Labeling 100 µg Protein5655752 Labelings1310
Buccutite™ Rapid PE-Texas Red Tandem Antibody Labeling KitOptimized for Labeling 25 µg Protein5656002 Labelings1343
Buccutite™ Rapid PE-Texas Red Tandem Antibody Labeling KitOptimized for Labeling 100 µg Protein5656002 Labelings1318
Buccutite™ Rapid APC Antibody Labeling KitOptimized for Labeling 25 µg Protein6516622 Labelings1313
Buccutite™ Rapid APC Antibody Labeling KitOptimized for Labeling 100 µg Protein6516622 Labelings1311
Buccutite™ Rapid PE-Cy5 Tandem Antibody Labeling KitOptimized for Labeling 25 µg Protein5656742 Labelings1340
Buccutite™ Rapid PE-Cy5 Tandem Antibody Labeling KitOptimized for Labeling 100 µg Protein5656742 Labelings1322
Buccutite™ Rapid PerCP Antibody Labeling KitOptimized for Labeling 25 µg Protein4826772 Labelings1353
Buccutite™ Rapid PerCP Antibody Labeling KitOptimized for Labeling 100 µg Protein4826772 Labelings1325

 

Additional Resources



Colorimetric, Fluorimetric and Chemiluminescent HRP Substrates


On it's own HRP conjugates have little value, as they lack the capacity to produce a measurable signal. To facilitate target visualization HRP conjugates require compounds known as substrates. In the presence of hydrogen peroxide, HRP catalyzes the oxidation of these substrates to produce a quantifiable signal. HRP substrates are commonly divided into three categories according to they type of singal it produces, these include, colorimetric, fluorimetric or chemiluminescent substrates.
  • Colorimetric substrates produce a visible color-change that can be measured using an absorbance microplate reader.
  • Fluorimetric substrates produce a highly fluorescent byproducts that can be measured using any fluorescence instrument. Offers greater sensitivity then colorimetric substrates.
  • Chemiluminescent substrates generate luminescent byproducts that can be measured using a luminometer. Since the emission of a photon is a result of a chemical reaction and not light excitation, there is minimal background interference.
 

Table 3. Colorimetric, fluorimetric and chemiluminescent HRP Substrates.

Substrate
Detection
Absorbance (nm)
Ex (nm)
Em (nm)
Unit size
Cat No.
ABTSColorimetric420 nm--100 mL
1 L
11013
11001
TMBColorimetric450 nm / Yellow
650 nm / Blue
--100 mL
1 L
11012
11003
Amplite® BlueFluorimetric-324 nm409 nm25 mg11005
Amplite® ADHPFluorimetric-570 nm583 nm25 mg11000
Amplite® RedFluorimetric-570 nm583 nm1000 Assays11011
Amplite® IRFluorimetric-646 nm667 nm1 mg11009
LuminolChemiluminescent--410 nm1 mg11050