Western blot analysis is a common approach in molecular and cell biology. It allows us to detect the specific proteins in a tissue homogenization or a tissue extract. However, if someone wants their western blot detection experiment to succeed, it is very important to learn the proper steps to prepare a sample correctly. It plays an important part in determining the quality of the result. It is a well-known fact that a good sample in any biological experiment makes the entire process much easier, further eliminating the risk of any errors.
What is Western blot analysis?
Western blot analysis is a method of detecting a specific protein in the samples of tissue extracts. This experiment involves the process such as protein preparation for western blot and dilution of the protein samples. Furthermore, the next step includes extraction and separation of proteins depending on their molecular weight. Electrophoresis is one such procedure that is generally used to separate proteins, especially at the initial stages. An electric current is passed through a medium like a polyacrylamide gel. In the end of electrophoresis and as part of overall western blot processing the proteins are transferred from the gel to a membrane, which create a replica of the protein separation pattern.
After the separation and the completed samples are transferred to a membrane which is blocked to avoid faulty results. If it is not done, the antibodies that are generally attached to the membrane make the detection of the presence of the target protein a gruesome task that may result in a false positive and high background. Therefore, once the protein has been immobilized and the membrane was blocked, one can focus on the primary antibody specific to the protein of interest. It can be detected using specific tags attached to a primary or secondary antibody.
How to prepare a sample
Now let us look at some of the things to keep in mind while preparing a sample for western blot analysis.
1. Choosing the Right Lysis Buffer for Your Sample
A lysis buffer is a buffer solution that is very useful in doing experiments involving molecular biology. While performing stripping in western blot, lysis buffer plays a crucial role as it aids in extracting the protein of interest. Therefore selecting a proper lysis buffer solution is the first and foremost thing to consider. If the wrong lysis buffer is used, it will make the extraction of the protein of interest a troublesome task.
Proteins must be isolated from cells and tissues using lysis buffer preparation for protein extraction, which is essential in diluting protein for western blot and performing a good western blot test. Lysis breaks down the cell membrane and helps in separating proteins from the cell’s non-soluble components.
Choosing a lysis buffer and calculating a suitable volume is usually a trial and error procedure. Lysis buffers range from mild with no detergent to strong, denaturing buffers with numerous detergents.
2. Incorporate Pro tease and Phosphate Inhibitors.
Pro tease inhibitors and phosphate inhibitors must be added during cell lysis to protect the proteins from enzymes generated during sample preparation. They restrict the rapid electrolysis and phosphorylation of the targets. It is generally advised to prepare the buffers before the experiment and keep it along with the cell maintaining 4 degrees Celsius throughout the procedure to preserve the protein.
3. Identify the Protein Concentration
It is important to determine the protein concentration of each lysate and equalize it across all samples to guarantee that the gel that being used is loaded evenly. The detection of protein levels and expression differences would be more reliable if the loading were equal in western blotting. Protein concentration can be determined using various protein assay methods, such as the Lowery assay, the BCA assay, the Bradford assay.
4. Prepare the Proteins
The protein sample must be diluted in the gel loading buffer. While Diluting protein for western blot, it is good to keep in mind that the protein extract is not diluted too much as it can result in inaccurate results. It can also aid in western blot detection. Chemicals like SDS, methanol, and Glycerol are generally found in the buffer. These chemicals are generally considered denaturing or reducing chemicals. As a result, diluting protein for western blot needs to be supervised. It is advised to use chemicals like Glycerol and Phenomenology blue. These chemicals aid in the buffer process.
Western blot stripping should also be done precisely for the detection the blot with a different primary antibodies in the next western blot analysis. Furthermore, no matter how thoroughly a process is generally implemented, the process is quite complicated, and there are many trial and errors to get the perfect sample. It is advised to create multiple samples at once. Keeping these things in mind can help conduct a successful western blot analysis.
