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.
| Cat. # | Size | Qty. | Price |
|---|---|---|---|
| 80283S | 100 µl |
|
| REACTIVITY | H M R Mk |
| SENSITIVITY | Endogenous |
| MW (kDa) | 45-48 |
| Source/Isotype | Rabbit IgG |
Product Information
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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: 263
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly142 of human ALKBH5 protein.
N6-methyladenosine (m6A) is an abundant and reversible RNA modification that plays an important role in mRNA splicing, processing, and stability. Demethylation of m6A is carried out by two enzymes, fat mass and obesity-related protein (FTO) and AlkB homolog 5 (ALKBH5). ALKBH5 belongs to the AlkB subfamily of Fe(II) and 2-oxoglutarate-dependent dioxygenases, and specifically recognizes the m6A modification within a conserved binding pocket (1). ALKBH5 knockout mice have been shown to be viable but harbor defects in spermatogenesis, primarily due to rampant apoptosis in spermatocytes (2). Specifically, ALKBH5 is required for late meiotic and haploid phases of spermatogenesis, and loss of m6A removal impairs mRNA splicing and stability necessary for the expression of key meiotic genes (3). ALKBH5 expression has been shown to be elevated in hypoxic models of breast cancer, resulting in increased m6A demethylation of NANOG mRNA and consequently promoting increased NANOG protein expression and accumulation of breast cancer stem cells (4). Knockdown of ALKBH5 has been shown to impair tumor formation in MDA-MB-231 breast cancer cells, and inhibition of ALKBH5 also represses tumorigenesis in glioblastoma stem-like cells (4-5). Interestingly, ALKBH5 may also show promise as a novel biomarker for pancreatic cancer, where increased expression was associated with higher overall survival rates (6).
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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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