Why western blots

It is also important to note that not all primary antibodies are suitable for western blotting and the application should be verified, if possible, before purchasing a new primary antibody. In general, the primary antibody that recognizes the target protein in a western blot is not directly detectable. Therefore, tagged secondary antibodies are used as the means of ultimately detecting the target antigen indirect detection. A wide variety of labeled secondary antibodies can be used for western blot detection.

The choice of secondary antibody depends on either the species of animal in which the primary antibody was raised the host species or any tag linked to the primary antibody e. For example, if the primary antibody is an unmodified mouse monoclonal antibody, then the secondary antibody must be an anti-mouse IgG secondary or non-IgG antibody obtained from a non-mouse host. However, the optimal dilution of a given antibody with a particular detection system must be determined experimentally.

More sensitive detection systems require less antibody than lower sensitivity systems and can result in substantial savings on antibody costs and allow a limited supply of antibody to be stretched out over more experiments. Using lower amounts of antibody can also have the added benefit of reduced background because the limited amount of antibody shows increased specificity for the target with the highest affinity.

Antibody dilutions are typically made in the wash buffer. The presence of detergent and a small amount of the blocking agent in the antibody diluent often helps to minimize background, thereby increasing the signal-to-noise ratio.

Conversely, adding too much blocking agent or detergent to the antibody dilution solution can prevent efficient binding of the antibody to the antigen, causing reduced signal as well as reduced background. While there are many different tags that can be conjugated to a secondary or primary antibody, the detection method used will limit the choice of what can be used in a western blotting assay.

Radioisotopes were used extensively in the past, but they are expensive, have a short shelf-life, offer no improvement in signal-to-noise ratio and require special handling and disposal. Alternative labels are enzymes and fluorophores. Enzymatic labels are most commonly used for western blotting and, although they require extra steps, can be extremely sensitive when optimized with an appropriate substrate.

Horseradish peroxidase HRP , and to a lesser extent, alkaline phosphatase AP are the two enzymes used most extensively as labels for protein detection. An array of chromogenic , fluorogenic , and chemiluminescent substrates are available for use with either enzyme.

Alkaline phosphatase offers a distinct advantage over other enzymes in that its reaction rate remains linear, improving sensitivity by simply allowing a reaction to proceed for a longer time period.

Unfortunately, the increased reaction time often leads to high background signal resulting in low signal-to-noise ratios. Horseradish peroxidase—conjugated antibodies are considered superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody due the smaller size of HRP enzyme and compatibility with conjugation reactions. In addition, the high activity rate, good stability, low cost, and wide availability of substrates make HRP the enzyme of choice for most applications.

Enzyme-conjugated antibodies offer the most flexibility in detection and documentation methods for western blotting because of the variety of substrates available. While not as sensitive as other substrates, chromogenic substrates allow direct visualization of signal development.

Unfortunately, chromogenic substrates tend to fade as the blot dries or during storage, making the blot itself an unreliable means of documentation. However, it is fairly straightforward to either photocopy or directly scan the blot in order to make a permanent replica of chromogenic western blot results. Chemiluminescent blotting substrates differ from other substrates in that the signal is a transient product of the enzyme-substrate reaction and persists only as long as the reaction is occurring.

If either the substrate is used up or the enzyme loses activity, then the reaction will cease and signal will be lost. However, in well-optimized assays using proper antibody dilutions and sufficient substrate, the reaction can produce stable output of light for 1 to 24 hours depending on the substrate, allowing consistent and sensitive detection that may be documented with X-ray film or digital imaging equipment. While X-ray film can be used to obtain semi-quantitative data, digital imaging is more sensitive because of the broad dynamic range of detection, allowing researchers to obtain quantitative data from western blots.

The use of fluorophore-conjugated antibodies requires fewer steps because there is no substrate development step in the assay. While the protocol is shorter, this method requires special equipment in order to detect and document the fluorescent signal due to the need for an excitation light source. Recent advances in digital imaging and the development of newer generation fluorophores such as infrared, near-infrared, and quantum dots has increased the sensitivity and popularity of using fluorescent probes for western blotting and other immunoassays.

Although the equipment and fluorophore-conjugated antibodies can be quite expensive, this method has the added advantage of multiplex compatibility using more than one fluorophore in the same experiment.

In addition, chemical waste is further reduced compared to other blotting procedures. Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Search All. Overview of Western Blotting. See Navigation. What is a western blot? Page contents Introduction Electrophoretic separation of proteins Transferring proteins to a membrane Blocking nonspecific sites Wash buffer formulations Primary and secondary antibodies Detection methods.

Direct method Indirect method Advantages Requires only one antibody Eliminates problems with secondary antibody cross-reactivity Disadvantages Label may interfere with target binding Potential for high background if antibody specificity for target is weak Conjugated primary antibodies may be costly Selection of conjugated primary antibodies may be limited Advantages Signal amplification by secondary antibody Vast selection of conjugated secondary antibodies One secondary antibody may be used with a number of different primary antibodies Use of secondary antibody does not inhibit primary antibody target binding Use of labeled secondary antibodies provides options for multiple detection methods Disadvantages Nonspecific staining may increase background Additional steps are required when using indirect method.

Electrophoretic separation of proteins. Transferring proteins to a membrane. Towbin, et al. PNAS — Next, the membrane goes through a treatment called blocking, which prevents any nonspecific reactions from occurring.

The membrane is then incubated with an antibody called the primary antibody, which specifically binds to the protein of interest. Following incubation, any unbound primary antibody is washed away, and the membrane is incubated yet again, but this time with a secondary antibody that specifically recognizes and binds to the primary antibody.

The secondary antibody is linked to a reporter enzyme that produces color or light, which allows it to be easily detected and imaged.

These steps permit a specific protein to be detected from among a mixture of proteins. Western blot. Further Exploration Concept Links for further exploration protein peptide amino acid gel electrophoresis biotechnology proteome. Related Concepts 6. For example, if each sample is a protein mixture of cells that are in different phases of the cell cycle, then western blotting will reveal how much a protein is present or absent during each phase.

Many proteins are cleaved in order to be activated or have naturally occurring truncation isoforms. Each isoform may have a different level of activity, a different target protein, or represent a different cellular state. Western blotting is great for detecting the ratio of truncated to normal isoforms of a protein. Some proteins are engineered, through the process of molecular cloning, to contain short sequences of amino acids that serve as a tag.

Common tags include the HA-tag and the Myc-tag. These tags serve as a foreign protein epitope that does not naturally occur in the biological system being studied.

Thus, the tag makes the protein easy to detect compared to all other naturally occurring proteins. An antibody directed to the tag will identify the presence and amount of the tagged protein in the western blot.

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