Proteins are macromolecules consisting of long chains of amino acids. They include enzymes, cell signaling proteins, ligand binding proteins, and cytoskeletal proteins. Protein extraction is the first step for most proteomics research methods (e.g. Western Blot, SDS-PAGE, mass spectrometry) as well as protein purification. Many techniques have been developed to maximize yield and purity for samples such as tissues or cultured cells. To get a high protein yield, samples (e.g. plant and animal tissues, cultured cells) must be homogenized uniformly and efficiently. This can be challenging as many samples contain protease protein-degrading enzymes as well as other interfering substances (e.g. keratins, phenolics, lipids, and organic acids). Thanks to 3D bead-beating technology — the gold standard for mechanical disruption — the Precellys homogenizers can homogenize virtually any type of tissues or cells and ensure that researchers always get the highest protein yield and purity.
Below, our top scientists have shared best practices for homogenization with the Precellys products range to maximize the recovery of high-quality proteins.
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Tips for a successful protein extraction
Choice of buffer and ratio mg tissue/volume of lysis buffer
The composition of the homogenization buffer should be adjusted according to the sample type, the location of the protein of interest, the required yield, and the nature of the downstream analysis. In general, a RIPA buffer can be a good starting point for optimization (150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 50 mM Tris-HCl (pH 7.4)). Depending on the assays, detergents such as SDS can be added to the buffer to improve the recovery of soluble protein.
Depending on downstream application the addition phosphatase and proteases inhibitors is advised to prevent protease-mediated protein degradation and/or dephosphorylation. Bertin’s Life Sciences team has developed the Protein safe kits, a state-of-the-art lysing kit containing a proprietary mix of protease inhibitors (see Figure 2). It should be noted that for some applications such as mass spectrometry, protease inhibitors should be used with great caution as they can interfere with results. We recommend using our standard lysing kits rather than the Protein Safe kits in the case of mass spectrometry workflows.
Another important factor is finding a good ratio (mg tissue/µl lysis buffer) between the quantity of tissue to homogenize and the volume of lysis buffer added in the homogenization tube. Overloading tissue in the tube could lead to poor or incomplete homogenization. On the other hand, adding too much lysis buffer will result in over-diluted non-exploitable samples. Depending on the tissue sample you are working on, a good starting point for the tissue/buffer ratio can be found in the table below.
Proteins are thermosensitive molecules, and as such, should be protected against excessive heating during homogenization. Consequently, protocols combining adapted homogenization speeds with short homogenization time (<15s) and lower cycles should be preferred. The Cryolys Evolution allows a precise control of samples temperature during each Precellys run.