When extracting native proteins from tissue samples, researchers must identify a method that both achieves consistency and maximizes the levels of detectable target. Here, we explain the rationale behind native protein extraction and look at some of the methods involved. We also share practical tips for safeguarding sample quality.
While native proteins are often studied using immunohistochemical staining methods, their extraction from tissue samples enables many other research techniques. For example, potential biomarkers can be identified using antibody microarrays, protein-protein interactions can be investigated via immunoprecipitation and native Western blot, and protein expression under conditions of health and disease can readily be compared with either singleplex or multiplex ELISAs. In addition, native mass spectrometry is an emerging technique for characterizing proteins in their non-denatured states. With native mass spectrometry, researchers can measure a protein’s thermodynamic and kinetic properties, determine the stoichiometry of protein complexes, resolve post-translational modifications such as phosphorylation or glycosylation, and examine ternary and quaternary structure.
For tissue samples to be analyzed using the methods just described, they must be processed from a solid form into a solution. This typically begins with cutting the tissue sample into several smaller pieces using a scalpel to ensure even, efficient homogenization—an umbrella term for the various methods used to disrupt tissue samples and release intracellular contents of interest. Next, the homogenized material is centrifuged to remove any unwanted cellular debris and the supernatant is directly analyzed. Alternatively, the sample may undergo further processing to isolate specific components such as intraorganellar targets or DNA binding proteins.
Homogenization methods can broadly be categorized as manual or mechanical. Manual methods involve grinding the tissue by hand, such as with a Dounce homogenizer (a device comprising a cylindrical glass container and two fixed diameter pestles, each providing different levels of shearing) or a pellet pestle (for disrupting smaller samples contained in microtubes). Compared to mechanical homogenization, manual techniques are relatively gentle. Mechanical homogenization methods include the use of sonicators, which exploit high frequency sound waves for sample disruption; bead mills, which are based on high-speed shaking of samples with glass or stainless steel beads; and blenders, which use rotating blades to disperse the tissue.
When deciding how to extract a native protein, it is important to identify a method that will maximize the levels of detectable target without compromising its utility for downstream research. Often, manual homogenization is preferred since mechanical methods can quickly heat up the sample, even when performed on ice. However, manual homogenization may be impractical when dealing with large sample numbers.
The amount of tissue required is another key consideration, especially if multiple tests are to be performed on the resultant extract. As a general rule, researchers can expect to recover around 10-fold less protein than the original weight of tissue used, making it critical to plan biopsies accordingly.
A further factor to consider is the lysis buffer. While a non-denaturing RIPA buffer is suitable for most tissue types, researchers may need to optimize the buffer formulation for solubilizing certain target proteins. Lysis buffers should always include protease and phosphatase inhibitors to prevent cellular proteases from digesting other proteins (including phosphoproteins) within the sample, and should be prepared fresh on the day of the assay to ensure they are free from contamination and inhibitors are performing optimally.
For biological assays to generate consistent results, it is recommended that the amount of tissue being processed is similar across samples to minimize variation during the homogenization process. Tissues should be weighed carefully, and be rinsed with phosphate buffered saline (PBS) prior to homogenization to remove any fluids that could contaminate the extract.
Once the tissue is homogenized, the proteins will begin to denature and dephosphorylate almost immediately. Although protease inhibitors will go some way toward protecting against this, it is essential to keep samples and buffers on ice and maintain homogenization apparatus at low temperatures. Extracts should be aliquoted and frozen straight away and multiple freeze thaw cycles avoided whenever possible.
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