Protein extraction, or cell lysis, is the foundational procedure used to release a cell’s contents for scientific analysis. Since most proteins function inside the cell, researchers must physically or chemically break open cellular barriers to access intracellular components without destroying the proteins. The resulting complex solution, known as the cell lysate, contains all the cellular proteins, nucleic acids, and small molecules needed for subsequent experiments.
Preparing the Cellular Sample
Before the cell membrane is ruptured, the biological sample must be prepared to ensure the final protein extract is clean. For cultured cells, preparation begins with harvesting, where cells are detached using a scraper or pelleted from the growth medium by low-speed centrifugation (typically 500 to 1000 \(\times\) g). The culture medium must be completely removed because serum proteins within it contaminate the final sample and interfere with accurate measurement.
Following harvest, the cell pellet is washed multiple times with an ice-cold balanced salt solution, such as Phosphate-Buffered Saline (PBS), to eliminate residual medium components. Maintaining a low temperature throughout this preparation phase is paramount, often by keeping all reagents and samples on ice or at \(4^\circ\text{C}\). This practice, known as maintaining the “cold chain,” slows the activity of destructive enzymes released once the cell is broken open.
Methods for Disrupting the Cell Membrane
The method chosen to disrupt the cell membrane depends heavily on the cell type and its structural rigidity. Animal cells are relatively easy to lyse due to their simple plasma membrane, but bacterial, yeast, and plant cells require more aggressive techniques because of their robust cell walls.
Mechanical Disruption
Mechanical methods use physical force to sheer the cell open. Sonication employs high-frequency sound waves that generate intense pressure changes, causing the cell membrane to rupture. Homogenization forces the cell suspension through a narrow space under high pressure or uses a tight-fitting pestle to shear the membranes. For tissue samples, manual grinding or pulverization following liquid nitrogen freezing is often necessary to break the tough fibrous structure.
Chemical Disruption
Chemical methods rely on specialized lysis buffers containing detergents to dissolve the lipid bilayer of the cell membrane. Detergents disrupt the interaction between lipids, effectively solubilizing the membrane and releasing the proteins. Non-ionic detergents (e.g., Triton X-100 or NP-40) are milder and used when preserving the protein’s natural structure is important. Ionic detergents, such as Sodium Dodecyl Sulfate (SDS), are highly effective at denaturing and solubilizing all proteins, including those tightly bound to membranes.
Enzymatic and Osmotic Methods
Enzymatic methods are often used in combination with other techniques, especially for cells with thick cell walls. For example, the enzyme lysozyme degrades the peptidoglycan layer of bacterial cell walls, making the cells more susceptible to lysis. A final, gentler method is osmotic shock, where cells are rapidly moved from a high-salt to a low-salt solution, causing water to rush in and burst the membrane due to internal pressure.
Stabilizing the Extract and Removing Debris
Immediately after cell disruption, destructive enzymes are released from cellular compartments. Lysis buffers are formulated to stabilize the proteins and maintain their function. A buffer system, such as Tris-HCl, provides a stable pH environment, while salts like sodium chloride maintain a physiological ionic strength to prevent protein aggregation.
The most important stabilizing agents are protease and phosphatase inhibitors, which are added to the lysis buffer just before use. Protease inhibitors block proteases, enzymes that rapidly degrade proteins by cleaving peptide bonds. Phosphatase inhibitors prevent the removal of phosphate groups from proteins, a modification linked to signaling pathways.
After stabilization, the crude lysate must be clarified to separate the soluble protein fraction from insoluble cellular debris. This clarification is accomplished through centrifugation, typically at 10,000 to 20,000 \(\times\) g for 10 to 20 minutes at \(4^\circ\text{C}\). Insoluble material, including cell wall fragments and nuclei, forms a dense pellet at the bottom of the tube. The clarified solution, called the supernatant, contains the desired soluble proteins and is transferred for analysis.
Measuring and Storing the Final Protein Solution
Before any downstream analysis, the total protein concentration in the final clarified solution must be quantified. This step ensures that equal amounts of protein are used in subsequent experiments, such as Western blotting, allowing for valid comparison of protein levels between different samples.
Colorimetric assays are the most common quantification methods, relying on a chemical reaction that produces a detectable color change proportional to the amount of protein present. The Bicinchoninic Acid (BCA) assay and the Bradford assay are widely used, with the choice depending on compatibility with the detergents in the lysis buffer.
For long-term preservation, the protein extract is divided into small portions, or aliquots, and immediately frozen, usually at \(-80^\circ\text{C}\). Aliquoting the sample is a practice that minimizes the number of freeze-thaw cycles any single portion undergoes. Repeated freezing and thawing can cause protein degradation and aggregation, reducing the integrity and function of the sample over time.