Western blotting, also known as immunoblotting, is a well-established and widely used technique for the detection and analysis of proteins. The method is based on building an antibody:protein complex via specific binding of antibodies to proteins immobilized on a membrane and detecting the bound antibody with one of several detection methods.
Although the details of Western blotting protocols may vary from among applications, with adaptations to suit specific protein characteristics and the level of information required, they all follow some common basic steps. In this guide, we’ll explore these seven steps to Western blot.
Did you know: First described in 1979, the method of Western blotting has since become one of the most commonly used methods in life science research.
Step 1. Sample Preparation:
The process begins with the sample of interest usually undergoing some degree of preliminary treatment before continuing to separation by electrophoresis. Biological matrices are complex. The target protein is likely to be one among many thousands present in the sample, in addition to nucleic acids, polysaccharides, and lipids, all of which might interfere with the analysis.
Numerous methods are available for disrupting cells and preparing their contents for analysis by Western blotting. Use extraction procedures that are as mild as possible. Extract proteins quickly, on ice, if possible, in the presence of a suitable buffer to maintain pH, ionic strength, and stability in order to prevent protein degradation. It is not usually necessary to treat samples prior to 1-D gel electrophoresis. However, sample cleanup improves performance by removing potentially interfering compounds such as nucleic acids, polysaccharides, and salts. Our SDS-PAGE Clean-Up Kit is designed for the preparation of samples that are difficult to analyze due to the presence of salts or a low protein concentration.
Step 2. Gel Electrophoresis:
Electrophoresis is the second step,step and is a commonly used method for separating proteins based on size, shape, and/or charge. Electrophoresis is a separation technique based on the mobility of charged molecules in an electric field. It is used mainly for the analysis and purification of large molecules such as proteins or nucleic acids. This then aids the selection process when considering the conditions that will enable you to most effectively get the most information you require from your specific analysis.
Electrophoresis is normally carried out by loading a sample containing the molecules of interest into a well in a porous matrix (polyacrylamide) to which a voltage is then applied. Differently sized, shaped, and charged molecules in the sample move through the matrix at different velocities. At the end of the separation, the molecules are detected as bands at different positions in the matrix.
When selecting a gel, it is important to use an acrylamide concentration that will allow optimal separation of the proteins in your sample. High molecular weight proteins will be optimally resolved in gels containing a lower acrylamide content, while smaller proteins should ideally be run in more acrylamide-dense gels. The image shows the separation pattern for nine different proteins for each acrylamide concentration. Molecular weight markers such as the Amersham ECL™ Rainbow markers can used to define the size of proteins run in a gel.
Step 3. Transfer:
On completion of protein separation by polyacrylamide gel electrophoresis (PAGE), the next step is to transfer the proteins from the gel to a solid support membrane, usually made of a chemically inert substance, such as nitrocellulose or PVDF. Blotting makes it possible to detect the proteins on the membrane using specific antibodies. The proteins transferred from the gels are immobilized at their respective relative migration positions at the time when the electric current of the gel run was stopped. Both Amersham Protran™ NC and Hybond™ PVDF membranes offer high protein binding capacity ideal for use in Western blotting.
Step 4. Antibody Probing:
Once your protein samples are separated and transferred onto a membrane, the protein of interest is detected and localized using a specific antibody. Usually, Western blotting protocols utilize an unlabeled primary antibody directed against the target protein and a species-specific, labeled secondary antibody directed against the constant region of the primary antibody. The secondary antibody serves not only as a carrier of the label but is also a mechanism to amplify the emitted signals, as many secondary antibodies can theoretically bind simultaneously to the primary antibody. This is one of the most effective ways to maximize the potential sensitivity of the assay.
For this reason, secondary antibodies are most often polyclonal and can target epitopes on the framework regions of the primary antibody; specificity is thus limited to species and immunoglobulin isotype. The signal emitted by the labeled secondary antibody is then measured and is proportional to the quantity of protein of interest present on the membrane. Blocking with BSA or Amersham ECL™ blocking reagent is an important first step before antibody probing to avoid nonspecific binding of antibodies to the membranes. Secondary antibodies are usually either conjugated to CyDye™ for fluorescent detection or to horseradish peroxidase, such as the Amersham ECL™ HRP conjugated antibodies for chemiluminescent based detection.
Step 5. Detection:
Radioisotopic and chromogenic reagents have been widely used for many years but have declined in popularity due to safety issues with handling radioactive isotopes and poor sensitivity with chromogenic reagents. Chemiluminescence and fluorescence are now the two most commonly used detection methods in western blotting. Radioisotopic and chromogenic reagents have been widely used for many years, but have declined in popularity due to safety issues with handling radioactive isotopes and poor sensitivity with chromogenic reagents. Enzymatic detection methods, such as chemiluminescence require the addition of a reagent that emits light when it reacts with an enzyme (HRP) conjugated to a secondary antibody. Considerable efforts have been made to develop HRP-based detection reagents such as Amersham ECL™ Prime and ECL Select™ so as to obtain higher detection sensitivity, stronger light intensity, and long-lasting signals.
Fluorescence-based detection, using on the other hand, requires no additional reagents after binding of the labeled secondary antibody. Fluorescence-based detection systems use a fluorescent entity, or fluorophore, directly conjugated to an antibody or streptavidin. The fluorophore can be excited using a light source of a specific wavelength causing light emission.
Instead of adding a detection reagent, fluorescent signals can be directly detected with equipment, such as laser scanners, fitted with suitable light sources and emission filters. Amersham ECL Plex™ uses Cy™3 and Cy™3- based detection. while Amersham CyDye™ 700 and 800 uses NIR based detection which is gaining popularity in recent years. Fluorescent based detection also enables multiplexing where more than one protein targets can be detected on the same blot. Fluorescent based detection also makes it easier to carry out total protein normalization using Amersham™ Quickstain.
Step 6. Imaging:
The last step in the Western blotting workflow before data analysis is image capture. Enhanced chemiluminescence (ECL™) is based on the reaction between an added luminol substrate and horseradish peroxidase (HRP)-labeled antibodies. In the presence of HRP, hydrogen peroxide catalysescatalyzes the oxidation of luminol, a reaction that results in the emission of light. The chemiluminescent light signal can then be detected on X-ray film or by digital imaging with a charge-coupled device (CCD) camera-based imager such as the Amersham ImageQuant™ 800.
When using fluorescence detection, a fluorophore is conjugated to the primary or secondary antibody. Light is emitted by the fluorophore after excitation via a specific wavelength of light. This can be detected by CCD based imagers such as the Amersham ImageQuant™ 800 or with highly sensitive laser scanners such as the Amersham Typhoon™.
Detection of signals using the Amersham ImageQuant™ 800 CCD imager or the Typhoon™ laser scanner results in one or more visible protein bands on the membrane image. The molecular weight of the protein can be estimated by comparison with marker proteins and the amount of protein can be determined as this is related to band intensity (within the limits of the detection system).
In most applications, it is enough to confirm protein presence and roughly estimate the amount. However, other applications demand a quantitative analysis that defines protein levels in either relative or absolute terms. For this the ImageQuant™ TL software offers the full range of analysis tools needed to accurately quantify proteins on the blot. The IQTL software is also available for regulated environments (IQTL SecurITy) to enable data traceability.