What is PVDF used for?
As western blot is a modification of the Southern blots for the detection and qualitative and quantitative analysis of proteins in a protein mixture. The process consists of several sub-steps:
1. Preparation of the sample
2. Gel electrophoresis
3. Transfer of the proteins to a membrane (blotting)
4. Blockage of the membrane
5. staining (optional)
6. Detection reaction or visualization
Before the actual blotting, the sample used must first be prepared for the procedure. As a rule, tissue samples, blood or similarly complex mixtures are used as the starting preparation, which contain other components in addition to the proteins to be examined. These must first be separated from the proteins. Some target structures are also located inside cells or even in individual cell organelles, which is why the cell membrane has to be ruptured. Both can usually be achieved through a combination of washing and lysing with a buffer solution that is specially tailored to the target protein. It is important that the buffer solutions contain specific protease and phosphatase inhibitors, which largely prevent the enzymatic cleavage of the proteins during lysis. Denaturation can also be significantly reduced by keeping the temperature of the sample below 4 ° C.
In some cases, however, it can make sense to deliberately denature a few target proteins so that they can then be recognized by specific antibodies. In this way, the proteins can be extracted reliably and sent for further processing. The proteins are then transferred from the solution to a gel in gel electrophoresis.
After the proteins have been separated from the other components of the preparation used in gel electrophoresis, the transfer of the fragments to a suitable carrier membrane, while maintaining the fragment pattern, is the central step of the western blottings represent.
The gel is stretched together with the membrane on absorbent paper and sponges in a cassette and immersed in a buffer solution. There are three different types of membrane available, which differ in their properties and are better or less suitable depending on the purpose of the blotting: polyvinylidene fluoride (PVDF), nitrocellulose and nylon.
For the western blot PVDF is often used because it offers a higher sensitivity than the other materials due to its high protein binding capacity (170-200µg / cm2). This results from the property that PVDF binds proteins in two ways: hydrophobic and via a dipole bond. Due to the high sensitivity, however, there is a stronger background coloration of the membrane, which can make subsequent analysis more difficult.
Further advantages compared to nitrocellulose and nylon are the easier handling, since PVDF is tear-resistant, and the lack of need to use methanol in the buffer solution during the transfer. Methanol acts as an organic solvent and unintentional precipitation can occur. In the process, methanol interacts with hydrogen bonds and impairs intramolecular, hydrophobic interactions in the proteins, partially unfolding them and provoking a loss of activity.
The pore size also plays an important role, which can be selected accordingly for all commercially available membranes (0.1, 0.2 or 0.45 µm). Depending on the mesh size, different proteins can be targeted, which differ in size, mass, charge and shape.
There are two main systems for transferring the protein pattern from the gel to the membrane: wet blotting and semi-dry blotting. The main differences between the two systems are the different amounts of buffer solution used and the time required.
The wet blotting is the most widespread because the efficiency for the detection of proteins with low molecular weight is very high. In principle, the inclusion of air bubbles between the gel plate and the membrane should be avoided, otherwise inaccuracies in the protein quantification due to incomplete transfer can occur. At the wet blotting can do this very easily by deleting the so-called Blot sandwich To be taken into account. As Blot sandwich This is a construction in which the gel is clamped together with the membrane and sometimes several layers of filter paper soaked with buffer solution.
It is a cassette that prevents the components from slipping and compresses the structure evenly to facilitate signal transmission. The cassette is then placed vertically in a box and completely covered with running buffer (e.g. Tris-Tricine-Gel or Tris-GlycineGel). The running buffer contains up to 20% methanol. At this concentration there is no loss of activity of the proteins, but their affinity for the membrane is increased because the bond to SDS is loosened. This should deliver higher transfer rates.
A current of 30V or 1A is then applied to the box and the proteins migrate through the running buffer in the direction of the positive electrode, analogous to gel electrophoresis. On their way, they are intercepted by the transfer membrane and thus transferred in the exact position, with the duration of the transfer process fluctuating between 60 minutes and several hours. To avoid overheating the gel, simple ice packs are often used, or the blotting is carried out in a cool room.
However, compared to the semi-dry blotting, the second variant, a higher time and technical effort, which must be taken into account when selecting the method.
At the semi-dry blotting will that Blot sandwich stretched horizontally between the electrodes. Because the amount of running buffer used in this case is only so large that the Blot sandwich is completely soaked inside the cassette, the duration of the transfer amounts to 10-60 minutes under installation of 10-24 V or 0.1-0.4 A. Another time saving comes from the fact that the test setup is less complex than with wet blotting.
With both variants, depending on the proteins transferred, a characteristic band arises on the gel, which must now be fixed and made visible.
Optionally, the gel can now be stained to demonstrate the efficiency of the transfer. Various alternatives are available for staining, such as Coomassie, Ponceau-S staining, colloidal gold and fluorophores based on rare earth chelates.
In the next step, the membrane is blocked if the proteins are to be detected later using specific antibodies. The reason for this is that antibodies are also proteins and when applied to the membrane, due to their high affinity, would bind to free areas that are not already occupied with proteins by the blotting. The bound proteins are not affected if the blot was completely dried before blocking. The membrane is then rehydrated with blocking reagents such as defatted dry milk, bovine serum albumin or gelatin in order to saturate all free areas and to avoid non-specific binding of the antibodies later used as a probe.
The most individual step of the western blottings is the detection reaction with specific antibodies, for which a large number of protocols exist. The basic procedure, however, hardly differs, so that a basic protocol can be drawn up: If the membrane has been treated with blocking reagents, one can now make use of the fact that the antigen function of the proteins was not impaired during the previous work steps to detect the target protein. It is therefore possible to apply specific antibodies to the membrane, which then bind to the target protein and thus highlight it. These antibodies are called primary antibodies. A few washing steps remove weakly adhering, unspecifically bound antibodies from the membrane. However, the resulting immune complex is not visible. For visualization it is necessary that a secondary antibody binds to the primary antibody. This secondary antibody is directed against constant areas of the primary antibody and is conjugated to enzymes, radionuclides, fluorescent dyes or the like. The use of a secondary antibody also has the advantage that the signal is amplified because several secondary antibodies can bind to one primary antibody. This ensures a high level of efficiency. Which option you choose ultimately depends on the desired sensitivity and, last but not least, on the financial leeway.
In the last step, the blot is analyzed. The presence of the target protein is now verified and a qualitative and quantitative assessment can then be carried out.
After immunodetection, stripping can be used to remove the bound antibodies and make the membrane accessible for further analyzes.
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