An Introduction to Western Blotting

Western blotting is a technique used to detect specific proteins from samples such as cell extracts or tissue homogenates, also analyze recombinant proteins synthesized in vitro. Western Blot can also be used to identify a target protein on the basis of the special affinity between a certain antigen and its relative antibody. Western Blot generally contains three main steps, SDS-PAGE firstly, then blot of samples, and finally immunology test. Qualitative and semi-quantitative data can be acquired about the target protein through Western Blot. In the field of protein, western blot is widely used for the test of protein expression level.

Principle of Western Blot:

• Western Blot is an immunoblotting technique which rely on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules.
• In Western Blot, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein.
• The SDS-PAGE technique is a prerequisite for Western Blot.

Advantages and disadvantages of Western Blot

Advantages Disadvantages
• Sensitive test
• Nice, clean easily interpretable output
• Thousands of commercially available antibodies
• Tests denatured proteins, so changes in functional states can be detected (i.e., receptor phosphorylation, acetylation, methylation, etc)
• Not quick, slightly labor-intensive
• Tissue must be homogenized

Featured antibodies for your Western Blot

Elite rabbit monoclonal antibody (EliteRmab®) for Western Blot

Sino Biological has built up an effective rabbit monoclonal antibody production platform based on our unparalleled expertise in phage display antibody library technology, including the generation of rabbit antibody libraries and selection of high affinity antigen-specific antibodies by phage display.

What are advantages of rabbit monoclonal antibody? Monoclonal antibodies are traditionally produced by hybridoma - myeloma cells fused with the spleen cells, from a mouse which has acquired immunization of the specific antigen. It is cost-effective and easy to operate to use a mouse as a host to produce monoclonal antibodies. However, researchers have been confronted with a problem of mouse monoclonal antibodies - limitations. Firstly, many target proteins are highly conservative between mice and humans, and therefore, those proteins may be recognized as self-antigens by a mouse host, making the antigens less immunogenic than expected. Secondly, compared with rabbit monoclonal antibodies, mouse monoclonal antibodies have lower affinities, and thus have a weak attraction to bind to the target proteins.

Secondary antibody

A secondary antibody is an antibody that binds to a primary antibody or antibody fragments. Secondary antibodies which are marked as probes are useful for the detection, purification or cell sorting applications. Secondary antibodies may be monoclonal or polyclonal. They are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions. Specific secondary antibodies are usually used to work in specific laboratory applications. Frequently, any one of several secondary antibodies has a particular application adequately. They are chosen according to the source of the primary antibody, the class of the primary antibody (e.g., IgM or IgG), and the kind of label which is preferred. The optimal secondary antibody is normally identified through trial and error. Secondary antibodies have been very useful for many biochemical assays, including immunocytochemistry, ELISA, western blot, immunohistochemistry, and so on.

Loading control antibodies for Western Blot

Loading controls are usually proteins that exhibit high-level, constitutive expression in the cell type or sample you are examining. This ensures constant expression levels. Thus "housekeeping genes" are frequently chosen for use as loading controls. It is also important that the protein chosen as a loading control has a different molecular weight than the protein of interest so that the bands are distinct and expression levels quantifiable. Popular loading control detection antibodies include anti-β-Actin monoclonal or polyclonal antibodies, anti-COX-4, anti-GAPDH, anti-Tubulin and anti-VDAC/Porin antibodies.

Tag antibodies for Western Blot

Tag antibodies make it easy to detect expression of proteins with specific tags, Myc, GST, GFP, His, HA, etc. Besides, some tag antibodies can be used in purification of tag fusion proteins. Sino Biological offers quality tag antibodies for your research needs with affordable price, 30-80% cost saving compared with currently leading brands; and big discount for bulk order.

Tag antibodies are widely used in various applications, either in detection of protein expression, or cellular localization, and protein purification. Sino Biological provides high quality tag antibodies validated in many immunoassays, Western Blot, ELISA, etc.

Isotype control antibodies for Western Blot

Isotype Control IgG is essential for ELISA, Western Blot (WB), Immunohistochemistry (IHC) and Immunoprecipitation (IP) experiments. It's purpose is to estimate that the proteins stained in the experiment result are due to the specific interaction with the antibody. Some people use specific primary antibody alone. This does not consider of those proteins that may bind to regions of the immunoglobulin distinct from the specific antigen binding sites. This Non-Specific binding is due to Fc receptor binding or other protein-protein interactions.The best way is to use isotype-matched control for the experiment. The IgG isotyping control should have the same immunoglobulin subtype and be used at the same concentration as the specific detection antibody. Sino Biological has a very good selection of control IgGs and the quality is excellent.