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.
Immunohistochemical analysis of paraffin-embedded A20 syngeneic tumor using CD74 (F3R3L) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded MC-38 syngeneic tumor using CD74 (F3R3L) Rabbit mAb.
| Cat. # | Size | Qty. | Price |
|---|---|---|---|
| 82174S | 100 µl |
|
| REACTIVITY | M |
| SENSITIVITY | Endogenous |
| MW (kDa) | 31 |
| Source/Isotype | Rabbit IgG |
Product Information
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
| Immunohistochemistry (Paraffin) | 1:400 - 1:1600 |
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
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
|
RECOMMENDED DETECTION REAGENTS |
SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 | SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653 |
|---|---|---|
|
COMPATIBLE CHROMOGEN |
SignalStain® DAB Substrate Kit #8059 | SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713 |
| SignalStain® Vivid Purple Peroxidase Substrate Kit #96632 | SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit #12824 | |
| SignalStain® Deep Black Peroxidase Substrate Kit #72986 | ||
| SignalStain® Radiant Yellow Peroxidase Substrate Kit #69644 |
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted February 2010
revised June 2020
Protocol Id: 283
Mouse
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro138 of mouse CD74 protein.
CD74, which is also known as the MHC Class II-associated invariant chain (Ii), is a type II transmembrane glycoprotein that plays a critical role in the antigen presentation process as a chaperone of MHC Class II proteins. It is expressed at high levels on B cells and to a lesser extent on numerous antigen presenting cell (APC) types including dendritic cells, Langerhans cells, monocytes, and macrophages as well as non-traditional APCs such as epithelial cells (1,2). CD74 was initially identified for its ability to regulate the folding and intracellular trafficking of newly synthesized MHC Class II molecules. Following expression, CD74 self-assembles as a trimer that serves as a scaffold for the assembly of MHC Class II molecules. Through this interaction, CD74 blocks the peptide binding cleft of MHC Class II molecules and prevents their premature association with endogenous polypeptides (3). Binding to CD74 also facilitates the translocation of MHC Class II molecules from the endoplasmic reticulum to the endocytic compartments during antigen presentation (4). In addition to its role as an MHC Class II chaperone, CD74 is also the receptor for macrophage migration-inhibitory factor (MIF). Binding to CD74 and its co-receptor, CD44, has been shown to induce the activation of the NFkB and ERK pathways to promote cell proliferation and survival signals (5,6). Recent studies have identified CXCR2 and CXCR4 as co-receptors for CD74 where MIF binding to CD74 complexes contributes to MIF-mediated monocyte chemotaxis and the induction of Akt signaling, respectively (7,8). Increased CD74 surface expression has been reported under inflammatory conditions and in certain types of cancer cells implying a potential role in tumorigenesis (9).
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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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