Western blotting analysis, also known as immunoblotting or protein blotting, is a core analysis technique in cell and molecular biology. In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells. Western blots are in wide use across a broad range of scientific and clinical disciplines. Their ability to clearly show the presence of a specific protein both by size and through the binding of an antibody makes them well-suited for evaluating levels of protein expression in cells, and for monitoring fractions during protein purification.
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After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns reveal protein are only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is repeated for a structural protein, such as actin or tubulin, that should not change between samples. The amount of target protein is indexed to the structural protein to control between groups. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.
Colorimetric Detection
The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the nitrocellulose membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through densitometry (how intense the stain is) or spectrophotometry.
Chemiluminescence
Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which captures a digital image of the western blot. The image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used. So-called “enhanced chemiluminescent” (ECL) detection is considered to be among the most sensitive detection methods for blotting analysis.
Radioactive Detection
Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest (see image to the right). The importance of radioactive detection methods is declining [citation needed], because it is very expensive, health and safety risks are high and ECL provides a useful alternative.
Fluorescent Detection
The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photo sensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.
Secondary Antibody
Tag Antibody
Loading Control Antibody
Isotype Control Antibody
Immunological Detection of Antigens
Handling of Antibody and Antibody-Enzyme Conjugate Reagents
Reagent Preparation
Immunoblotting is typically used to determine the amount (dot blot) and molecular weight (western blot) of an antigen present in a complex mixture. The highly sensitive procedure below is suggested. All incubations are at room temperature.
This procedure is for nitrocellulose or PVDF membranes. Do not touch the membrane! Wear gloves. PVDF membranes must be pre-wet with 100% methanol before equilibrating in transfer buffer. Nylon membranes may be used, however, they are more difficult to block. To block nylon membranes use buffer without Tween-20, replace BSA with 10% nonfat dry milk and block for several hours to overnight at 4 degrees C. When selecting membranes, the specifications of the particular imaging systems with their available filters must be taken into consideration. Some membranes may exhibit auto fluorescence at certain wavelengths.
1) Western Blot Buffer Preparation
2) Western Blot Sample Preparation
3) Western Blot Gel Electrophoresis
4) Western Blot Transfer
5) Western Blot Blocking
6) Western Blot Detection
The Western blotting procedure relies upon three key elements to accomplish this task:
1) the separation of protein mixtures by size using gel electrophoresis
2) the efficient transfer of separated proteins to a solid support and
3) the specific detection of a target protein by appropriately matched antibodies
Once detected, the target protein will be visualized as a band on a blotting membrane, X-ray film, or an imaging system. Since Western blotting is accomplished rapidly, using simple equipment and inexpensive reagents, it is one of the most common laboratory techniques. The results achieved are also easy to interpret, unique, and unambiguous. Therefore, it is routinely used on its own, or along with other immunoassays, in research and clinical settings.
A typical Western-transfer protocol includes the following steps:
1) The proteins in the tested solution are separated into distinct bands by SDS-PAGE,
2) The size-separated proteins are transferred from the polyacrylamide gel to a nitrocellulose membrane (or another suitable matrix).
3) The membrane containing the protein bands is serially incubated with:
a) a suitable blocking reagent to prevent non-specific protein binding
b) a wash solution to rinse any unbound blocking reagent
c) a probing antibody (anti-protein-X antibody) that forms a specific immune complex with Protein-X
d) additional wash solution to remove any unbound antibody,
e) an enzyme-linked antibody that binds specifically to the Fc region of the anti-Protein-X antibody
f) additional wash solution to remove any unbound enzyme-linked antibody and finally
g) a substrate solution, which in the presence of the enzyme, yields an insoluble, colored product that precipitates at the site of the immune complex, thereby rendering the Protein X band visible.
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