Useful Websites for Western Blot

1.European Bioinformatics Institute

2.BIOSCI/Bionet News


4.National Academy of Sciences

5.Protocol Online

6.National Center for Biotechnology Information

7.Departments – University of South Carolina School of Medicine

8.PLOS Pathogens

9.Molecular Biology Protocols | Bioinformatics


Antibody Function

Activated B cells differentiate into either antibody-producing cells called plasma cells that secrete soluble antibody or memory cells that survive in the body for years afterward in order to allow the immune system to remember the antigens and respond faster upon future exposures.

At the prenatal and neonatal stages of life, the presence of antibodies is provided by passive immunization from the mother. Early endogenous antibody production varies for different kinds of antibodies, and usually appear in the first years of life.

Since antibodies exist freely in the bloodstream, they are part of the humoral immune system.

Circulating antibodies are produced by clonal B cells that specifically respond to only one antigen (an example is a virus capsid protein fragment).

Antibodies contribute to immunity in three ways: they prevent pathogens from entering or damaging cells by binding to them; they stimulate removal of pathogens by macrophages and other cells by coating the pathogen; and they trigger destruction of pathogens by stimulating other immune responses such as the complement pathway.

Activation of complement
Antibodies that bind to surface antigens on, for example, a bacterium attract the first component of the complement cascade with their Fc region and initiate activation of the “classical” complement system.

Activation of effector cells
To combat pathogens that replicate outside cells, antibodies bind to pathogens to link them together, causing them to agglutinate. Since an antibody has at least two paratopes it can bind more than one antigen by binding identical epitopes carried on the surfaces of these antigens.

By coating the pathogen, antibodies stimulate effector functions against the pathogen in cells that recognize their Fc region.

Rejection of xenotransplantated organs is thought to be, in part, the result of natural antibodies circulating in the serum of the recipient binding to α-Gal antigens expressed on the donor tissue.

Antibody Structure

Antibodies are heavy globular plasma proteins. They have sugar chains added to some of their amino acid residues. In other words, antibodies are “glycoproteins”.

The basic functional unit of each antibody is an immunoglobulin (Ig) monomer (containing only one Ig unit); secreted antibody can also be dimeric with two Ig units as with IgA, tetrameric with four Ig units like teleost fish IgM, or pentameric with five Ig units, like mammalian IgM.

Immunoglobulin domains
The Ig monomer is a “Y”-shaped molecule that consists of four polypeptide chains; two identical “heavy chains” and two identical ”light chains” connected by disulfide bonds.

They have a characteristic immunoglobulin fold in which two beta sheets create a “sandwich” shape, held together by interactions between conserved cysteines and other charged amino acids.

Heavy chain
There are five types of mammalian Ig heavy chain denoted by the Greek letters: α, δ, ε, γ, and μ.

The paratope is shaped at the amino terminal end of the antibody monomer by the variable domains from the heavy and light chains. The variable domain is also referred to as the FV region and is the most important region for binding to antigens.

More specifically, variable loops of β-strands, three each on the light (VL) and heavy (VH) chains are responsible for binding to the antigen. These loops are referred to as the complementarity determining regions (CDRs).

In the framework of the immune network theory, CDRs are also called idiotypes. According to immune network theory, the adaptive immune system is regulated by interactions between idiotypes.

The base of the Y plays a role in modulating immune cell activity. This region is called the ”Fc (Fragment, crystallizable) region”, and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody.

Antibody Forms

Considering antibody form, immunoglobulin should be firstly researched. Surface immunoglobulin (Ig) is attached to the membrane of the effector B cell by its transmembrane region, while antibodies are the secreted form of Ig and lack the trans-membrane region so that antibodies can be secreted into the bloodstream and body cavities.

As a result, surface Ig and antibodies are identical except for the transmembrane regions. Therefore, they are considered two forms of antibodies: soluble antibodies form or membrane-bound antibodies form.

The membrane-bound form of an antibody may be called a “surface immunoglobulin” (sIg) or a “membrane immunoglobulin” (mIg). It is part of the “B cell receptor” (BCR), which allows a B cell to detect when a specific antigen is present in the body and triggers B cell activation.

The BCR is composed of surface-bound IgD or IgM antibodies and associated Ig-α and Ig-β heterodimers, which are capable of signal transduction.

A typical human B cell will have 50,000 to 100,000 antibodies bound to its surface. In humans, the cell surface is bare around the B cell receptors for several thousand angstroms, which further isolates the BCRs from competing influences.


Western Blot Antibody Selection

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 Blotting

What Do You Know about Your Protein?

In order to make informed choices among the bewildering range of available transfer and detection methods, it is best to have as clear an idea as possible of your own particular requirements. In large part these choices will depend on the nature of your target protein. Even limited knowledge can be used to advantage.

How abundant is your protein? It isn’t necessary to answer the question in rigorously quantitative terms: an educated guess is sufficient. Are your samples easy to obtain and plentiful, or limited and precious? Is the sample likely to be rich in target protein (e.g., if the protein is overexpressed) or poor in target (perhaps a cytokine)? Obviously low protein concentration or severely limited sample size would require a more sensitive detection method.

What is the molecular weight of your target protein? Low MW proteins (12 kDa or less) are retained less efficiently than higher molecular weight proteins. Membranes with a pore size of 0.1 or 0.2 micron are recommended for transfer of these smaller proteins, and PVDF will tend to retain more low MW protein than nitrocellulose. The ultimate lower limit for transfer is somewhere around 5kDa, although this depends largely on the protein’s shape and charge.

The transfer of high molecular weight proteins (more than 100kDa) can benefit from the addition of up to 0.1% SDS to the transfer buffer (Lissilour and Godinot, 1990). Transfer time can also be increased to ensure efficient transfer of high molecular weight proteins.

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Western Blot Medical Diagnosis Applications

In recent medical field, western blot has a wide range of applications in medical diagnosis, such as the application of medical diagnosis for HIV infection, BSE, FIV, Hepatitis B Virus infection, and so on.

1. Western blot is applied in a confirmatory HIV-test to detect anti-HIV antibody in a human serum sample. Proteins like gp41, gp120, from known HIV-infected cells are separated and blotted on a membrane. Then, in the primary antibody incubation step, the serum to be tested is applied; free antibody is washed away, and a secondary anti-human antibody conjugated with an enzyme signal is added. Then the stained bands will indicate whether the patient’s serum contains anti-HIV antibody. This is the main principle of western blot medical diagnosis assay for HIV infection.

2. Under appropriate conditions, the western immunoblotting technique is quantitative. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separated viral proteins .Viral proteins are transferred quantitatively to nitrocellulose by electroblotting in SDS-containing buffer. Monoclonal antibodies directed against previously defined epitopes on the viral proteins were used as probes to detect viral protein synthesis and expression, as well as processing in animal tissues. Because of their polypeptide specificities, circulating polyclonal antibodies were also probed and characterized. Finally, a highly sensitive dot immunoblotting assay can analyze the sensitivity and denaturation of various epitopes on the viral proteins. Picogram quantities of viral antigens and antibodies were detected by this assay.

3. Western blot application in the definitive test for Bovine Spongiform Encephalopathy (also known as “mad cow disease”). Anti-PrPC antibody (T1) was obtained by immuning the rabbits with bovine prion protein (BoPrPC) expressed in E. coli. Because pathological prion protein (PrPSC) was resistant to protease treatment, extracts of brain tissue were digested with proteinase K and detected by western blot with T1 antibody. The results showed that protease-resistant pathological PrPSC was existed in golden hamster brain tissue which was inoculated with scrapie strain 263 K, but no protein existed in normal golden hamster brain homogenates which was detected with T1 antibody. Several bovines and sheep from Beijing were used for diagnosis of Bovine spongiform encephalopathy (BSE) and scrapie, their brain tissue were freshly collected and homogenated. The homogenates were separated on SDS-PAGE and detected by western blot with T1 antibody. The results indicated no protease-resistant protein(PrPSC) existed, this suggested they were not infected by BSE and scrapie. The same results were obtained with 1A8 antibody from England. These results indicated we could detect BSE and scrapie with T1 antibody.

4. Also, in veterinary medical field, western blot is sometimes applicated in the medical diagnosis for FIV in cats. The initial test was the ELISA. However, in ELISA, both anti-FIV and anti-FeLV antibodies can be tested at the same time, so there can be false positives with the ELISA test. An initial positive for FIV is followed up by a laboratory test, such as western blot test, which confirms that anti-FIV antibodies are present in the serum.

5. Medical diagnosis application of western blot in a confirmatory test for Hepatitis B infection. The genome of hepatitis B virus (HBV) is a partially double-stranded DNA molecule within virus particles that is approx 3.2 kbp long. It has four open reading frames, all on one strand, that encode the surface antigen or envelop polymerase, and the so-called “X” protein (HBxAg). Experiments and observations have suggested that HBxAg is a common marker in the livers of carriers. The ability to detect HBxAg may depend on the type of cell line used for expression. For example, HBxAg is detected by western blotting in transfected NIH3T3 cells.

6. Western blotting is also applied in some forms of Lyme disease diagnosis test. For medical tests like Lyme disease, western blotting is used in combination with another technique of ELISA (enzyme-linked immunosorbent assay). Since ELISA might sometimes yield false-positive results, western blotting works as a confirmation tool for the test result of ELISA.

1. Flisiak R, Wierzbicka I, Prokopowicz D. Western blot banding pattern in early Lyme borreliosis among patients from an endemic region of north-eastern Poland. Rocz Akad Med Bialymst. 1998;43:210-20.
2. Tylewska-Wierzbanowska S, Chmielewski T. Limitation of serological testing for Lyme borreliosis: evaluation of ELISA and western blot in comparison with PCR and culture methods. Wien Klin Wochenschr. 2002 Jul 31;114(13-14):601-5