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Recombinant antibodies offer several key advantages compared to traditional antibodies. These include superior lot-to-lot consistency, continuous supply, and animal-free manufacturing. As such, recombinant antibodies are seeing increased use for scientific research, especially as a means of addressing the ongoing reproducibility crisis.
Traditional polyclonal and monoclonal antibodies are the product of normal B cell development and genetic recombination. They are generated by immunizing an animal with an antigen to elicit an immune response. While polyclonal antibodies are secreted by many different B cell clones and recognize multiple antigenic epitopes, monoclonals originate from a single B cell clone and are specific for just one epitope.
Recombinant antibodies are monoclonal, but their production involves in vitro genetic manipulation. After cloning the antibody genes into an expression vector, this is then transfected into an appropriate host cell line for antibody expression. Mammalian cell lines are most commonly used for recombinant antibody production, although cell lines of bacterial, yeast, or insect origin are also suitable.
Because recombinant antibody production involves sequencing the antibody light and heavy chains, it is a highly controlled and reliable process. In contrast, hybridoma-based systems for producing monoclonal antibodies are subject to genetic drift and instability, increasing the potential for lot-to-lot variability or loss of antibody expression. Recombinant antibodies are highly consistent from lot to lot, thereby ensuring reproducible experimental results.
In vitro methods for producing antibodies are amenable to large-scale production, meaning antibody availability is unlikely to become a limiting factor. Moreover, since the recombinant antibody sequence is known, continuity of supply is assured; in situations where an antibody will be used to support large, long-term studies, this can be an especially critical factor.
Unlike traditional methods for antibody production, recombinant approaches avoid the need to use animals. Where polyclonal antibodies are purified directly from the serum of the immunized host, and monoclonals are purified from either hybridoma-derived tissue culture supernatant or ascites, recombinant antibodies are instead purified from the tissue culture supernatants of transfected host cell lines. Regardless of whether an antibody is polyclonal, monoclonal or recombinant, it must always be properly validated in the intended application prior to experimental use. At CST, we adhere to the Hallmarks of Antibody Validation™, six complementary strategies for determining the specificity, sensitivity, and functionality of an antibody in any given assay. By carefully tailoring these strategies to each antibody product, we guarantee that CST antibodies will work as expected, to help you achieve results you can trust.
| 製品# | サイズ | 数量 | 価格 | 在庫 |
|---|---|---|---|---|
| 38977S | 100 µl |
|
| REACTIVITY | H |
| SENSITIVITY | Endogenous |
| MW (kDa) | 187 |
| Source/Isotype | Rabbit IgG |
製品情報
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala1370 of human C3 protein.
Complement is a collection of soluble proteins secreted in the blood and other body fluids (1). As part of the innate immune system, complement proteins are involved in the clearance of pathogens and damaged cells in a process called opsonization, which results in the coating of a pathogen with antibodies and/or complement proteins to facilitate phagocytosis of debris or foreign pathogens. Complements are activated by a cascade of cleavage reactions, triggered initially by pattern recognition receptor-mediated detection of pathogens/debris. The cascade of cleavage-mediated products activates three distinct effector pathways, including inflammation, phagocytosis, and membrane attack, that represent a coordinated defense of the host organism. Several complement proteins are encoded in the mammalian genome, designated by the capital letter "C" followed by a number, in order by their discovery. Many complement cascades converge on C3, encoded by the C3 gene, by activating C3 convertase. The C3 gene generates a C3 polypeptide protein, composed of an α-subunit and a β-subunit linked by disulfide bonds. Activation of C3 convertase cleaves the C3 α-subunit to generate C3a, which acts as an anaphylatoxin to mediate the local inflammatory response. In addition to C3a, C3 convertase-mediated cleavage of C3 generates C3b. C3b has multiple functions and is a major effector molecule of the complement system. C3b binds to microbial surfaces to enable phagocytosis by phagocytic cells carrying the C3 receptor. C3b can also be further processed by serum proteases to generate other bioactive fragments. Finally, C3b can form complexes with other complement fragments to initiate cleavage of complement family members like C5, which initiates additional innate immune responses. In addition to the innate immune response, several components of the complement system, including C3, have been implicated in brain development, as well as neurodevelopmental and neurodegenerative diseases. The complement system plays a role in microglia-dependent synapse pruning of excess synapses during development, particularly in the visual system (2). Additionally, complement-mediated synaptic pruning may also contribute to neurodegenerative diseases, such as Alzheimer’s disease (3,4).
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.
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