The most critical section of western blot is the proper selection of western blot antibodies other than technical issues. The critical feature of primary antibody is the specificity for the epitope of your target protein. All steps of western blot must be optimized to minimize the non-specific background signals, and those include the determination of the most appropriate incubation condition for primary antibody during pre-experiment. The working dilution of an antigen affinity-purified polyclonal antibody is usually lower than that of a monoclonal antibody. To achieve a strong and specific signal, a high quality antibody that exhibits minimal cross-reactivity is supposed to be employed. Then how to find the suitable antibody, including the selection of primary antibody and the selection of secondary antibody; should monoclonal antibody be chosen or polyclonal antibody instead? For this question, you should be clear about the purpose or application of your western blot and explicit about the difference between monoclonal antibody and polyclonal antibody at first. In term of how to decide whether to use rabbit monoclonal antibody or mouse monoclonal antibody or others, the difference between rabbit MAbs and mouse MAbs should be considered, and then just choose the right one according to your western blot purpose. What about the selection of secondary antibody? What’s more, is there any principle or standard for western blot antibody selection? Which antibody supplier shall we turn to? Actually, there is usually more than one antibody available for the target protein. In order to narrow the span of western blot antibody selection, several aspects above must be clear and taken into consideration to select the appropriate antibody as far as possible.
- Primary Antibody Selection – Monoclonal Antibody versus Polyclonal Antibdoy
- Monoclonal Antibody Selection – Mouse MAbs versus Rabbit MAbs
- Secondary Antibody Selection
- Antibody Suppliers Selection – Antibody Suppliers
- Recommended Antibody Products
Primary Antibody Selection – Monoclonal Antibody versus Polyclonal Antibody
Before western blot, the primary antibody should be selected ahead of time. Then here comes the question about the selection of primary antibody. Should monoclonal antibody be chosen? Or polyclonal antibody might be a better choice? Whatever kind of primary antibody you choose, either monoclonal antibody or polyclonal antibody, a primary antibody should be chosen that has been raised against the same species of the protein of interest being studied. And it all depends on the intended use of the primary antibody and whether it is readily available from commercial supplier or researchers. Afterwards, in combination with the difference between monoclonal antibody and polyclonal antibody, make the final decision of selection of primary antibody.
PAbs can be generated with shorter period of production, with less cost and less technical skill than is required to produce MAbs. The production procedure of polyclonal antibody is much easier than the production procedure of monoclonal antibody. One can reasonably expect to obtain PAbs within one or two months, whereas the acquirement of hybridomas and subsequent production of MAbs can sometimes take up to a year or longer in some cases, therefore requiring more expense and more time-consuming.
The principal advantages of monoclonal antibody are the consistency and homogeneity. MAb can be under consideration owing to its monospecificity if the purpose of your western blot is evaluating changes in molecular conformation, protein-protein interactions, and phosphorylation states, or identifying single members of protein families. It also allows for the potential of structural analysis (e.g., x-ray crystallography or gene sequencing) to be determined for the antibody on a molecular level. However, the monospecificity of MAbs may also limit their application. Small changes in the epitope structure of your target protein (e.g., as a consequence of genetic polymorphism, glycosylation, and denaturation) can considerably affect the function of a MAb. In that case, MAbs should be generated to the state of the antigen to which it will eventually need to bind. In contrast, PAbs are heterogeneous and can recognize a couple of antigenic epitopes, so the structural change in a single or small number of epitopes can seldom make a difference to the affinity. PAbs are also more stable over a broad salt concentration and pH.
Another key advantage of MAbs is that once the expected hybridoma is obtained, MAbs can be generated as a constant and renewable resource. In contrast, PAbs generated to the same antigen using multiple animals will differ among immunized animals, and their avidity may change as they are harvested over time. The quantity of PAbs obtained is limited by the size of the animal and its lifespan. Actually, this the concern of antibody suppliers when they decide to choose which antibody they may concentrate to produce according to their own case.
However, note that PAbs often have better specificity than MAbs, because they are produced by a large number of B cell clones, each generating antibodies to a specific epitope, and polyclonal sera are a composite of antibodies with unique specificities.
Monoclonal Antibody Selection – Mouse MAbs versus Rabbit MAbs
If you are finally sure about a selection of monoclonal antibody as primary antibody, then it’s time to think about which monoclonal antibody you will choose, mouse monoclonal antibody or rabbit monoclonal antibody?
Monoclonal antibodies are traditionally obtained by hybridoma cells, a fusion of myeloma cells with spleen cells from a mouse that has been immunized with the desired antigen. Mouse monoclonal antibodies, the production of which takes a mouse as a host, is cost-effective and easily operated. However, limitations of mouse monoclonal antibodies have been found by researchers. First, there are many highly conserved proteins between mice and humans, which therefore, makes some target human proteins less immunogenic since the conserved human proteins can be falsely recognized by the host of mice as self-antigens. Second, mouse monoclonal antibodies have relatively low affinities, and thus have a weak attraction to bind to the target protein. These problems can be prevented when using rabbit as a host anima to produce monoclonal antibodies. The availability of rabbit monoclonal antibodies is highly desirable for their high specificity and affinity, and the immune response to mouse antigens is greatly improved. In addition, rabbits are known to produce antibodies to many antigens that are not especially immunogenic in mice. Rabbit monoclonal antibodies also have other advantages, such as more diverse epitope recognition and improved immune response to small-size epitopes, compared with mouse monoclonal antibodies. However, rabbit monoclonal antibodies are much more difficult to develop compared to mouse or rat monoclonal antibodies, due to lack of a stable fusion partner of cell line for hybridoma development, and are therefore usually a little more expensive.
Selection of Secondary Antibody
Most hosts of the production of primary antibodies are mice or rabbits; therefore, anti-mouse IgG and anti-rabbit IgG are the most popular types of secondary antibodies. Goat is the most easily and frequently used host by suppliers to produce polyclonal anti-mouse and anti-rabbit secondary antibodies. Consequently, goat secondary antibodies against mouse IgG and rabbit IgG are commercially available in the widest variety of forms. Several kinds of anti-mouse and anti-rabbit secondary antibodies from other host species are also available.
There are a wide variety of selection of labeled secondary antibodies for western blot. The selection of secondary antibody rely on the species of animal in which the primary antibody that is expected to be employed was raised (the host species). For example, if the primary antibody is a rabbit monoclonal antibody, an anti-rabbit IgG must be selected as the secondary antibody. The experiment will not often be affected by the host species of the secondary antibody. However, secondary antibodies can be obtained from many host species, and if a secondary antibody causes high background in a particular assay, another host species may be selected. Another means to reduce background is to select a secondary antibody that has been pre-adsorbed, such as serum proteins from other species. This pre-adsorption process removes antibodies that have the potential to produce cross-reactivity with serum proteins, including antibodies.
Antibody Suppliers Selection – Antibody Suppliers
There are so many antibody suppliers in the world, so eye-dazzling that researchers may feel a little perplexed for the right selection of antibody. As long as an antibody is finally selected, then those world-renowned antibody suppliers are the first choice for your order. However, a variety of world-renowned antibody suppliers in the world rely on their own sales agent to sell antibodies, which makes a hidden price, so, those antibody suppliers whose business model is direct sales through network are especially recommended since a buyer can have a direct communication with the antibody manufacturer for any detailed exact information about products and order at a more reasonable price. Besides, more attention should be paid to the case that whether the antibody to be ordered is suitable for western blot or / and ELISA, IP, IHC according to the products information. Also, focus on the antigen feature for the antibody, some antibody suppliers employ peptides as immunogens to produce antibodies, while some antibody suppliers use a full-length protein as an immunogen. Obviously, the quality of the antibody produced by full-length protein immunization is much higher. For example, almost all the antibodies from Pranovo Biotech are produced with highly purified recombinant proteins as immunogens, and it has no sales agent but directly sell products through network to the world, including many biotech companies and worldwide academic institutions, also world-famous medicine enterprises. At last, consider your own intended use; make a selection.
Western Blot 