The western blot is a widely used analytical technique used in molecular biology, immunogenetics and other molecular biology disciplines to detect specific. Western Blot (WB) is a common method to detect and analyze proteins. It is built on a technique that involves transferring, also known as blotting, proteins. Western blotting is an important technique used in cell and molecular biology. By using a western blot, researchers are able to identify specific proteins from a.
Okay, so while the concept of using electricity to transfer your proteins from your gel to a membrane is straightforward, some details are often overlooked. It is not a covalent interaction! So why is this forgettable detail important? As your best blot will always be the first probing , it is best to always probe for your weakest antibody first, as subsequent handling and stripping can unstick your proteins-of-interest. Before you start throwing antibody on your membrane, you need to prep it by coating all the unoccupied sticky portions of the membrane with a blocking agent.
There are a number of blocking agents to choose from, and even some buffers such as Tris supposedly stick to the membrane to facilitate the block. The two most commonly used blocking agents are milk and bovine serum albumin BSA.
While milk is the old standby, some antibodies nonspecifically stick to the milk, which causes dirty blots and in the process effectively dilute your antibody. Thus, because it has been through some purification steps, BSA is the preferred blocking agent when probing with phosphorylation sensitive antibodies. There is also a barnyard animal clause in blocking choice.
Instead you will need to block with something like chicken, rabbit or undergrad serum A joke!! And you never know which until you try it! Just remember the basic concepts of antibody recognition: And if you are lucky these epitopes are on your protein-of-interest!
Secondary antibody selection depends on your primary antibody and your detection system. Secondary antibodies recognize the heavy constant chain of your primary antibody and this is species -specific.
So pay attention to what species your primary antibody was made in! And what antibodies might be in your sample! As would be the case if you did immunoprecipitation. Luckily, some special secondary antibodies only recognize disulfide bonded primary antibodies, thereby eliminating the chance of seeing antibodies used previously in the experiment.
Secondary antibodies are conjugated to different developing molecules depending on the type of developing system you are using. For film you will likely use HRP horseradish peroxidase or AP alkaline phosphatase ; for laser capture, you will need a fluorophore. How you develop your Western blot depends on your developing system. In other words, what developing molecule is coupled to the heavy chain of your antibody or in some short-cut cases, your primary antibody.
If the HRP is conjugated to your secondary antibody, your membrane needs to be incubated with a luminol containing kit. The resulting chemiluminescence can then be captured on film in a dark room, or with a camera image detector. There are two methods for the blotting called wet and semi-dry. The wet conditions are preferred when the transfer must be efficient and give high quality regarding distinct and sharp bands.
In addition, this is the better choice for transfer of larger protein complex. The gel, membrane, and filter papers are completely immersed in buffer during the transfer and there is no risk of drying out the gel. Semi-dry blotting is more rapid and less volume of buffer is needed. However, this transfer method is usually less efficient, especially for larger proteins, and there is a risk of overheating and drying the gel when using extended transfer times.
The forth step of the WB is antibody probing. In order to prevent unspecific binding of the antibodies to the membrane, rather than binding specific to the protein of interest, a substance is used to block out the residual sites on the membrane. Milk is easy to get hold of and inexpensive, however not suitable for all detection labels.
Fish gelatin gives lower background but can mask some proteins as well as being a relatively expensive blocking buffer. BSA is inexpensive, whereas serum can contain immunoglobulins giving rise to cross-reactivity.
Careful selection of the blocking agent is key since none of the blocking buffers are ideal for all different antigen-antibody interactions. The blocking procedure consists of incubating the membrane in the appropriate blocking buffer for an hour or longer. Blocking is a delicate balance between reducing the background without decreasing the signal from the protein of interest. The blocked membrane is thereafter incubated with the primary antibody.
It is preferred to incubate the antibody with BSA if the antibody is going to be re-used. After washing the membrane, the membrane is incubated with the secondary antibody that binds to the primary antibody.
The secondary antibody is labeled with a reporter. When using a polyclonal antibody as secondary antibody, it may give rise to some background. In the case of background staining, the secondary antibody may be pre-blocked with non-immune serum from the host it was generated in. Optimization of the concentration of the secondary antibody is recommended due to quite extended variations between antibodies as well as detection system used.
In the fifth step of a WB, the protein-antibody-antibody complex is detected on the membrane. There are several kinds of labeling of the secondary antibody, e. Amongst enzymes the most common is HRP used together with chemiluminescent, chemifluorescent, or chromogenic substances.
HRP has a high substrate specificity giving low background, is stable, and inexpensive. In chemiluminescense the HRP enzyme catalyzes the oxidation of luminol from the luminol peroxide detection reagent. The multi-step reaction generates light emission. Certain chemicals like phenols can enhance the emitted light. A direct method is the use of fluorescence; the fluorophores emit light after being excited and no detection agent is needed. It is well suitable for quantitative Western and since different fluorophores emit light of different wavelengths it is possible to perform multiplexing and specific detection of more than one protein at the time.
To further enhance the signal intensity a two-step biotin streptavidin based system may be used. Gold conjugation is also a method where proteins stain dark red due to accumulation of gold. It is also possible to use radioisotopes but they require special handling and are quite expensive.
Imaging Imaging is the sixth step of WB and the capturing can be analogue using a film, or digitally preformed with a CCD camera or scanner capturing the different kinds of emitted signals. The CCD imaging device enables quantitation with high detection sensitivity and a broad linear range with no chemical waste or need for a dark room.
It may be used to detect membranes, stained gels, or for ultraviolet light applications. The last step of a WB is to analyze the results. In a typical qualitative application, the presence of a protein of interest is confirmed, the amount is approximated by visual inspection, and the size is determined by comparison with a marker. Improvements and developments, especially towards highly sensitive detection reagents and advanced imaging techniques, make WB a potential tool for quantitative analysis.
The quantitative applications entail a definition of the amount of protein in relative or absolute terms. Some factors are to take under consideration like sensitivity, signal stability, linear dynamic range, normalization, and the signal-to-noise ratio. The minimum of protein that can be seen in a given assay gives the limits of detection LOD , and the limit of signal intensity that can be reliably used for precise quantification is the limit of quantification LOQ.
Factors that affect these terms are antibody quality and concentrations as well as exposure times when considering the minimum amount of protein detected. A stable signal system expands the time window for reaching high sensitivity, multiple exposures, and possibility to detect weak bands.
The range that allows an even and precise quantitation where the signal intensity still is proportional to the amount of protein is called the linear dynamic range. It is important to avoid signal saturation due to excessive amounts of protein or high concentrations of antibodies. A low LOD and quantitation of both weak and strong signals gives a broad linear dynamic range. The protein of interest should be normalized to an internal reference that allows fluctuations in amount of protein loaded onto each well or different concentrations.
This can be achieved with housekeeping or spiked protein. The ratio between the signal and noise is important in order to properly quantitate the protein. This is of outmost importance when detecting weak bands where a higher background is expected. Although the theoretical weight of many proteins differ from the real weight due to post translational modifications, almost all commercial antibodies are validated using WB.
Within the Human Protein Atlas project WB is used for quality control of the polyclonal antibodies generated in the project.
After purification, the antibodies are used to detect bands in a setup of lysate and different tissues. The entire WB protocol, including dilution of both primary- and secondary antibodies and the final detection step, is performed in a routine manner and no specific experimental optimization is made for individual antibodies.
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Know Your Western Blot Jargon! A Quick Review.
Western blotting(also called protein immunoblotting because an antibody is used to specifically detect its antigen) is a widely accepted analytical technique used. A western blot experiment, or western blotting (also called immunoblotting, because an antibody is used to specifically detect its antigen) was introduced by . Western blotting, also known as immunoblotting or protein blotting, is a core technique in cell and molecular biology. In most basic terms, it is used to detect the.