Antibody Plasmid Collection
Addgene Receives a NIH BRAIN Initiative Grant to Create an Open-Access Recombinant Antibody Resource! Coming in early 2022.
Antibodies are produced by the immune system to help defend against invaders such as bacteria, viruses, or even perceived threats such as proteins in peanuts or plant pollen. They are large proteins that bind to an antigen, a particular part of a foreign substance. In addition to being useful for our bodies to fight off intruders, antibodies are also extremely useful to researchers in a lab. When an antibody binds tightly to a specific antigen, scientists can use it to pull out a protein of choice from a mix of other proteins, visualize a protein under a microscope, or detect when the protein is present in a sample--just to name a few.
A standard antibody is made up of 4 protein chains: two large heavy chains and two small light chains. These four domains come together to form a “Y” shape, as shown in the picture to the right. The two arms of the Y structure are responsible for binding the antigen, and are called the antigen binding fragments (Fab). The tips of the Fab fragments contain highly variable regions, shown in light blue and orange, that bind to a specific antigen, triggering an immune response. The other part of the antibody is the constant region, shown in dark blue and orange, which is the same within each class of antibody. Antibodies are divided into five major classes, IgM, IgG, IgA, IgD, and IgE, based on their constant region structure and immune function.
Traditional sources of antibodies for research purposes include animals or hybridoma cells, but are difficult to generate due to expense and time. An alternative approach is to use synthetically produced recombinant antibodies, created by cloning antibody components into plasmids and expressing these in bacteria, mammalian cells, yeast, plants, or insect cells. Benefits include consistency between lots and the ability to optimize the antibody’s antigen binding sequence to improve binding and reproducibility.
Some scientists choose to work with full length recombinant monoclonal antibodies (R-mAbs) expressed in mammalian cells. Monoclonal antibodies are derived from an individual clone targeting a single antigen. Examples of R-mAbs are the monoclonal antibodies (NeuroMabs) extensively validated for neuroscience research applications from the NeuroMab/Trimmer Lab Recombinant mAb Plasmid Collection. Once the plasmids are transiently transfected into mammalian tissue culture cells, the R-mAbs are secreted into the culture media and can be collected for use.
Other scientists may choose to work with a smaller fragment of an antibody, which could offer increased stability and ease of production due to their small size. These recombinant antibody fragments can be used in experiments such as immunoprecipitation and super-resolution microscopy. Two commonly used plasmid-based antibody fragments are described below:
ScFvs (single-chain variable fragment) include parts of the variable regions of heavy (VH) and light chains (VL) fused together to form a single polypeptide. Unlike full length antibodies, which are often produced in mammalian cell cultures, scFvs are often produced in bacterial cell cultures such as E. coli. While full-length antibodies generally do not fold properly in the cytoplasm, soluble scFv antibodies have been successfully expressed. The Vale lab has created several scFv fragments which bind to the GCN4 peptide, from the SunTag system, fused to sfGFP for imaging.
Nanobodies are small single chain antibodies that are derived from an unusual type of IgG antibody called a heavy chain antibody (HCab), which are unique to camels, llamas, alpacas and other camelids. In terms of structure, HCabs are like a pared down version of a standard IgG antibody. Their small size also allows better tissue penetration and decreases the distance between a fluorescent tag and the target antigen, which can lead to higher resolution for super-resolution microscopy. Examples include secondary antibodies from the Gorlich lab and GFP nanobodies from the Cepko lab.
Cloning and Expressing Antibodies
The following table highlights plasmid collections that can be used to create and express plasmid-based antibodies.
The table below highlights plasmids that feature expression of antibodies, nanobodies or ScFvs. Use the search bar or sort buttons to find antibody plasmids for:
- Common antigens such as GFP or mCherry
- Monocolonals, Nanobodies, Sybodies, or ScFVs
- Secondary antibodies (search for "anti-mouse" or "anti-rabbit")
Addgene Blog Posts
- Antibodies 101: Introduction to Antibodies
- Addgene Receives NIH BRAIN Initiative Grant to Create Open-Access Recombinant Antibody Resource
- RANbodies: Reporter Nanobody Fusions
- Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification
- Secondary Nanobody Toolbox
- Using Nanobodies to Create GFP Scaffolds for Transcriptional Activation
- Developmental Studies Hybridoma Bank: Over 25 Years of Antibody Sharing
- The Human Protein Atlas - Open access project that aims to map all the human proteins in cells, tissues, and organs.
- The Antibody Society - An international, non-profit association representing individuals and organizations involved in antibody-related research and development.
- Developmental Studies Hybridoma Bank - Sells monoclonal antibodies at cost to facilitate basic science.
- Geneva Antibody Facility - Provides the academic community with easy, affordable access to well-characterized rAbs to generate better and more reproducible data, in a collaborative way.
- The Antibody Registry - Gives researchers a way to universally identify antibodies used in their research.
- Neuromab - Provides a unique neuroscience-based approach to generating mouse monoclonal antibodies (mAbs) optimized for use in mammalian brain (NeuroMabs).
- NCI Office of Cancer Clinical Proteomics Research Antibody Portal - Provides access to a large number of standardized renewable affinity reagents (to cancer-associated targets) and accompanying characterization data.
- SAbDab - Database of antibody structures that updates on a weekly basis.