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 |
|---|---|---|---|
| 72172S | 100 µl |
|
| REACTIVITY | H |
| SENSITIVITY | Endogenous (IHC-P) |
| MW (kDa) | |
| Source/Isotype | Rabbit IgG |
Product Information
| Application | Dilution |
|---|---|
| IHC Leica Bond | 1:50 - 1:200 |
| Immunohistochemistry (Paraffin) | 1:200 |
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 a carrier-free (BSA and azide free) version of this product see product #99825
NOTE: Please see product datasheet or product webpage for appropriate antibody dilution^.
| Step | Reagents | Time/Temperature | |
|---|---|---|---|
| 1 | Dewax | BOND™ Dewax Solution, 100% Alcohol, BOND™ Wash Solution | Pre-programmed Leica® BOND™ |
| 2 | Antigen Retrieval | BOND™ Epitope Retrieval ER2 Solution | 20 min., 100˚C | Protocol: HIER 20 min with ER2 |
| 3 | Peroxide Block | Refine Detection Kit Peroxide Block* | 5 min. |
| WASH | BOND™ Wash Solution | 3x 0:00 min. | |
| 4 | Protein Block (optional) | #5425 NGS or #15019 Animal-Free Blocking Solution | 20 min. |
| 5 | Primary Antibody^ | Dilute in #8112 SignalStain® Antibody Diluent | 60 min. |
| WASH | BOND™ Wash Solution | 3x 2:00 min. | |
| NA | Post Primary Mouse Linker | Refine Detection Kit Post Primary* | Not Applied |
| 6 | Secondary Detection | Refine Detection Kit Polymer* | 10 min. |
| WASH | BOND™ Wash Solution/Deionized Water | Custom (see below) | |
| 7a | Visualization | Refine Detection Kit Mixed DAB Refine* | 0:00 min. |
| 7b | Visualization | Refine Detection Kit Mixed DAB Refine* | 10 min. |
| WASH | Deionized Water | 3x 0:00 min. | |
| 8 | Counterstain | Refine Detection Kit Hematoxylin* | 5 min. |
| WASH | Deionized Water | 0:00 min. | |
| WASH | BOND™ Wash Solution | 0:00 min. | |
| WASH | Deionized Water | 0:00 min. | |
| 9 | Dehydration (Offline): | ||
| Incubate sections in 95% ethanol two times for 10 seconds each. | |||
| Repeat in 100% ethanol, incubating sections two times for 10 seconds each. | |||
| Repeat in xylene, incubating sections two times for 10 seconds each. | |||
| 10 | Mount sections with coverslips and #14177 SignalStain® Mounting Medium | ||
| Optional Custom wash: | BOND™ Wash Solution | 2:00 | |
| BOND™ Wash Solution | Dispenser Type: OPEN 0:00 | ||
| BOND™ Wash Solution | 2:00 | ||
| BOND™ Wash Solution | Dispenser Type: OPEN 0:00 | ||
| BOND™ Wash Solution | 0:00 | ||
| Deionized Water | 0:00 | ||
*Reagent included in BOND™ Polymer Refine Detection Kit (Catalog No: DS9800)
LEICA® is a registered trademark of Leica Microsystems IR GmbH.
BOND™ is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.
posted August 2018
revised September 2018
Protocol Id: 1464
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
Human
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the extracellular domains of human CXCR5 protein.
CXCR5 is a G protein-coupled receptor belonging to the chemokine receptor subfamily (1). Upon binding of its ligand, the chemokine CXCL13, CXCR5 initiates multiple intracellular signaling pathways that regulate cell proliferation, survival, and migration (2). CXCR5 is expressed in both mature B cells and follicular helper T cells, and responds to CXCL13 gradient to control lymphocyte migration towards secondary lymphoid tissues (3). CXCR5 has also been shown to be highly expressed in primary breast tumors, in correlation with their propensity to grow and metastasize (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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