Antibodies and epitope tags
Loading Control Antibody Guide
Knockout-validated primary antibodies
Secondary antibodies resources
Alexa Fluor secondary antibodies
Biotinylated secondary antibodies
Gold-labelled secondary antibodies
How to prepare Phosphate Buffered Saline (PBS)
How to design a flow cytometry panel
Enhancing Detection of Low-Abundance Proteins
9 tips for detecting phosphorylation events using a Western Blot
Western Blotting with Tissue Lysates
Western blot membrane stripping protocol
Immunohistochemistry and Immunocytochemistry
Chromogenic and Fluorescent detection
Preparing paraffin-embedded and frozen samples for Immunohistochemistry
Competitive ELISA assay protocol
Measuring analyte concentration using serial dilutions and standard curve
Choosing the right antibody:
Antibodies are a type of glycoproteins, which bind to specific antigens and are synthesised by B-cells as a response to foreign molecule invasion in the body, and are made from four polypeptide chains, creating the well-known Y-unit.
Furthermore, each Y section consists of an identical pair of heavy chains and an identical pair of light chains, named after their length and sequence differences and connected by disulfide bonds. What differentiates one antibody from another is its variable region, located at the top of the Y section. This region is responsible for the tight and specific binding to the epitope of the antigen, that the immunoglobulin has been secreted against.
The most important part of this process is the interaction between the antibody and the antigen. If there is an issue with the quality or compatibility of the antibody, the results will not be as reliable or consistent compared to using high-grade validated products.
There are two main types of antibodies: Monoclonal and Polyclonal. Both have their advantages and disadvantages and suitable applications.
They are made by fusing antibody producing cells from the spleen of the immunized animal (usually a rat or mouse) with an immortalized cell line to produce single specificity antibodies that can be purified from tissue culture supernatant.
One of their biggest advantages in their long-term use, is that after the initial costs for production, they can create unlimited supply.
On the other hand, if other proteins share the same epitope, like isomers or common motifs, monoclonal antibodies tend to cross-react and are generally less sensitive due to having only a single molecule binding to the target.
Polyclonal antibodies consist of a mixed pool of immunoglobulin molecules that bind to several different epitopes found on a single antigen. Polyclonals are usually produced in rabbits, donkeys, sheep, and goats, and are purified from serum.
This type of antibodies has varying specificities to multiple epitopes, compared to monoclonal antibodies, which bind to a single epitope within a target antigen.
Furthermore, due to the several antibodies which bind to the same target, the sensitivity is much higher, and they can identify the whole target protein via binding at several different sites.
On the opposite side, they are cheaper to produce but in the long run, supply is greatly limited and there is higher variability between preparations.
In some cases, there are no antibodies on the target protein, which creates a problem with carrying out immunodetection techniques. In this case, epitope tags and matching tag antibodies are used to perform a Western blot for example.
This technique works by inserting a small DNA sequence for a familiar antigenic epitope while the protein is cloned. Due to already existing antibodies, which will bind to that specific epitope, the modified protein will be detected by the antibody. This technique helps greatly with carrying out immunodetection quickly and efficiently.
On the flip side, any alterations to the protein separate is from its original form, leading to possible differences between the two in terms of reactivity and binding.