- Western blotting (WB)
- Immunohistochemistry - Paraffin
- ELISA
- DotBlot
- ELISA Capture
- ELISA Detection
- ELISPOT
- Flow cytometry
- Immunohistochemistry
- Immunohistochemistry - Frozen
- Immunoprecipitation
MagicLink™ HRP Antibody Conjugation Kit
Catalog #: bp-50002
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Overview
HRP can be conjugated to antibody/protein by thiol-maleimide and hydrazone coupling approaches. The thiol-maleimide approach is commonly performed with a bi- functional linker (such as SMCC). However, SMCC-modified protein is extremely unstable and often self-reactive since it often contains both amine and thiol groups that cause a significant amount of homo-crosslinking. The conjugation linkage is exchangeable when other thiol groups are present. In the hydrazone coupling approach, the conjugation reaction is based on a reaction between hydrazine and aldehyde. This method is low yielding although a catalyst is used. The size of the molecule and inaccessibility of the functional group contribute to low yield. In addition, linkage stability is an issue.
BroadPharm's MagicLink™ technology solves all the problems by combining PEG and proprietary crosslinking chemistry. MagicLink™ chemistry and kit do not need TCEP, DTT, or catalysts at all!
First, antibody reacts with MAGIC NHS ester to yield the MAGIC activated antibody which instantly reacts with LINK preactivated HRP to yield antibody-HRP conjugate with a stable ring linkage.- Novelty: 3rd generation protein crosslink technology
- Yield: conjugation efficiency around 95-100%
- Rate: instant reaction; the whole process less than 1 hour
- Linkage stability: most stable covalent bonded conjugates on the market
- Consistence: good reproducibility from batch to batch
- Flexible: use your own HRP and antibody at your desired ratio
- Shelf life: long, stable freeze dried ship at ambient temperature
- Antibody selection: any proteins/antibody, antibody fragment
Post-conjugation clean-up step not necessary - HRP activity: > 250 U/mg
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Components
- MAGIC NHS (MW ~900)
- LINK activated HRP
- Reaction buffer
- Protein concentrator
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Application
MagicLink HRP antibody Labeling Kits are used to conjugate HRP with primary antibody, secondary antibodies, and other biomolecules for the bioassay below. -
Images
A typical flow chart of HRP -antibody conjugation by MagicLink technology
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Protocol
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Reference
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- Sternberger LA, Petrali JP.; The unlabeled antibody enzyme method. Attempted use of peroxidase-conjugated antigen as the third layer in the technique; J Histochem Cytochem (1977): 1036
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- Tijssen P, Kurstak E.; Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays; Anal Biochem (1984): 451
- Boorsma DM, Cuello AC, van Leeuwen FW.; Direct immunocytochemistry with a horseradish peroxidase-conjugated monoclonal antibody against substance; J Histochem Cytochem (1982): 1211
- Broorsma DM, Steefkerk JG, Kors N.; Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate; J Histochem Cytochem (1976): 1017
- Nakane PK, Kawaoi A.; Peroxidase-labeled antibody. A new method of conjugation; J Histochem Cytochem (1974): 1084
- Tresca JP, Ricoux R, Pontet M, Engler R.; Comparative activity of peroxidase-antibody conjugates with periodate and glutaraldehyde coupling according to an enzyme immunoassay; Ann Biol Clin (Paris) (1995): 227
- Presentini R, Terrana B.; Influence of the antibody-peroxidase coupling methods on the conjugate stability and on the methodologies for the preservation of the activity in time; J Immunoassay (1995): 309