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 |
|---|---|---|---|
| 78672S | 100 µl |
|
| REACTIVITY | H M R Mk |
| SENSITIVITY | Endogenous |
| MW (kDa) | 23 |
| 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 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, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding ubiquitylated Lys121 of human histone H2A.Z protein.
Modulation of chromatin structure plays a critical role in the regulation of transcription in eukaryotes. The nucleosome, made up of four core histone proteins (H2A, H2B, H3 and H4), is the primary building block of chromatin. In addition to the growing number of post-translational histone modifications regulating chromatin structure, cells can also exchange canonical histones with variant histones that can directly or indirectly modulate chromatin structure (1). There are five major variants of histone H2A: canonical H2A (most abundant), H2A.X, MacroH2A, H2ABbd and H2A.Z (2). Histone H2A.Z, the most conserved variant across species, functions as both a positive and negative regulator of transcription and is important for chromosome stability (2). Several homologous protein complexes, such as SWR-C (S. cerevisiae), TIP60 (D. melanogaster) and SRCAP (mammals), have been shown to catalyze the ATP-dependent exchange of H2A.Z for H2A in the nucleosome (3,4,5). This exchange of histone H2A variants changes histone-histone interactions in the nucleosome core and alters an acidic patch on the surface of the nucleosome, resulting in changes in nucleosome stability and binding of non-histone proteins such as HP1α (6,7).
H2A.Z is a histone H2A variant protein that is critical for proper regulation of gene expression. H2A.Z is localized throughout the genome, but appears to be most concentrated at the promoters and enhancers of active genes (8). Acetylation of histone H2A.Z at promoters and enhancers confers nucleosome destabilization and open chromatin confirmation, facilitating transcriptional activation (9-11). While the bulk of histone H2A.Z appears to be excluded from constitutive heterochromatin, histone H2A.Z is found in various forms of facultative heterochromatin, including the inactive X chromosome and transcriptionally poised bivalent gene promoters (8,12). In these heterochromatic regions of the genome, H2A.Z is mono-ubiquitylated on Lys120 and Lys121 by the Ring1B ubiquitin ligase found in the Polycomb Repressor Complex 1 (PRC1) (8,12). Mono-methylation of H2A.Z on Lys120 and Lys121 facilitates repression of gene expression by inhibiting the binding of the activating BRD4 protein and facilitating the recruitment of the Polycomb Repressor Complex 2 (PRC2), the latter of which methylates histone H3 on Lys27 and facilitates transcriptional repression (8).
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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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