Nitrocellulose is a crucial component of the western blot procedure, also known as protein immunblot. It is used to analyze the molecular weight of a particular protein in a given tissue sample, or to measure the amounts of protein it contains, says the Department of Biology at Davidson College. Nitrocellulose is used as blotting paper to retain the proteins once they have been separated from the rest of the sample.
About Western Blotting
A cell or tissue sample can contain 30,000 different proteins -- sometimes even more -- so this technique was developed to target the specific protein you want to analyze, says the Molecular Station website. The first step in the process is separating the proteins from the sample, then transferring them to a nitrocellulose membrane to stop them from degrading. You must use an antibody that is specific to the protein you want to identify to ensure it binds to the right protein so that it can then be detected.
Protein Separation
The process of separating the proteins in a given tissue sample is called electrophoresis, says westernblotting.org. Polyacrylamide gels are used to effectively strain the proteins according to size. The smaller proteins move through the gel first, while the largest are last to do so, causing the proteins to form size-specific bands in the gel. According to westernblotting.org, the most common method of electrophoresis used in western blotting is sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). This process uses an electric current to strain the proteins through the gel.
Proteins and Nitrocellulose
Once the proteins have been separated in the gel, they are transferred to a membrane, or blotting paper, of nitrocellulose, which helps preserve the proteins during the incubation and detection stages of the procedure, says the Department of Biology at Davidson College. More recently, polyvinylidene difluoride (PVDF) has been used as blotting paper because it is especially effective in maintaining protein retention in harsh acidic or basic conditions, says westernblotting.org.
Incubation and Antobodies
After you have transferred the proteins to the nitrocellulose membrane, you must incubate the preparation with non-fat dry milk, the proteins in which block other reactions, says westernblotting.org. At this stage, the protein-specific antibody is also added so that it can bind to the particular protein you are looking to identify, says the Molecular Station website.
Protein Detection
You will not be able to detect your protein, however, until you add a secondary antibody containing horse radish peroxidase enzyme. This substance causes a chemical reaction that results in light being released from the protein, which will show up as a colored spot on film, says the Molecular Station website. The number of spots that appear will help you determine the size and number of your target protein in the sample.
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