Lysate Preparation: How Do I Optimize My Extraction?
Technical tips to improve your sample preparation for Western blot
1. Cell lysis
For suspension cell culture samples, cells can be collected directly by centrifugation and washed with PBS or with saline 2–3 times to remove the serum in the medium. For adherent cells, though trypsin treatment is popular, we do not recommend it since trypsin can also digest the protein of interest. Instead, we recommend scraping off adherent cells when possible. Whether you are using suspension or adherent cells, the rest of the process is the same: lysis buffer is added, the sample is sonicated, and then centrifuged. Afterward, a small amount can be used to determine the concentration, with the remainder used for the WB.
2. Tissue lysate
Tissue protein extraction is more complex. The first and most critical step is to perform a clean dissection of the desired sample. Once the sample is dissected, it is necessary to wash the sample with PBS to remove blood contamination, thereby preventing nonspecific signal from a secondary antibody binding to the organism’s endogenous immunoglobulins. After washing, the sample is homogenized. Following homogenization, the general procedure is similar to that of the cell culture process. Unlike cell culture samples, however, the tissue is often rich in connective tissue, and some is difficult to dissolve in conventional lysates and might require experimentation to optimize results.
Preventing protein degradation
As mentioned earlier, many tissues and cells contain proteases. Here are some methods to mitigate these enzymes during sample preparation.
1. Use protease inhibitors.
Both PMSF and EDTA are inexpensive yet highly effective inhibitors, and are therefore used in almost all WB experiments.
2. Perform the procedure at a low temperature.
For the preparation of protein samples of common mammalian tissues or cells, all steps can be performed at low temperature, and all reagents should be precooled to reduce protease activity and prevent protein degradation. In particular, digestive system-related tissue samples should be processed as quickly as possible, and the preparation method should be grinding after flash freezing in liquid nitrogen to minimize sample degradation.
However, there are species such as zebrafish whose proteases are most active at low temperatures. In these cases, perform the procedure at a high temperature (50°C–60°C) where their activity is low.
3. When taking samples from multiple organs, order the dissections by protease activity.
Digestive system-related organs and macrophage-rich tissues (e.g., lungs) should be dissected and snap frozen first. Then, reproductive tissues should be processed. Heart, spleen, kidney, brain, and other organs can be dissected last.
Some cell lines, such as Raw 264.7 and U-937, have high protease activity. In these cases, consider using a high concentration of SDS to accelerate the extraction process.
Tissues and cells contain many other substances besides proteins and these can interfere with your WB. Here are some strategies to mitigate impurities in your sample:
1. Avoid cross-contamination.
During extraction, use sterile, clean tools, especially equipment such as homogenizers, grinding pestle, and dissection tools. Avoid using proteases to improve your protein extraction yield.
2. Sonicate the sample to remove nucleic acids.
Nucleic acids can bind to proteins and interfere with your analysis. If the prepared sample contains large amounts of nucleic acids, the target protein’s mobility may be affected, or the complex may form large aggregates that are insoluble. The solution is to break down nucleic acids using a sonicator, which renders them incapable of forming intact protein-binding domains.
3. Remove as much fatty tissue as possible from your dissected sample.
Like nucleic acids, lipids can bind to proteins and cause issues with your WB. If there is still some fatty tissue remaining after dissection, use of a silica column is recommended to adsorb lipids.
4. Maintain physiological salt ion concentration.
As mentioned earlier, high concentrations of salt ions may result in a smiley band. Additionally, if salt concentration among the different lanes is not uniform, the migration of the same protein in different lanes may be different. Therefore, in the sample preparation process, try to maintain physiological ion concentration across samples.
Fractionate or separate your sample to increase concentration of low-abundance proteins
Some proteins only reside in specific cells or organelles. Consequently, when using the whole lysate, its abundance may not reach the WB detection limit. In these cases, it is recommended to fractionate the desired cell subsets or specific organelles you need according to the literature.
Concentration leveling and evaluation
After protein extraction is complete, it is important to determine the protein concentration in your sample. A quantitative protein assay such as BCA will yield the most accurate results. It is also recommended to run SDS-PAGE to evaluate the quality of the lysate to ensure that the lysis is thorough and not degraded.
To ensure consistent sample loading, it is necessary to adjust the concentration so that it is consistent across your samples. Once this is completed, 4X loading buffer can be added to the sample.
Despite a variety of methods to ensure error-free protein extraction and similar concentrations, it is still strongly recommended to include loading controls in your experiments.
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