Cell disruption is a collection of techniques used for releasing biomolecules of interest from inside the cell. This blog will discuss the various techniques which will be broken down into two categories, gentle and harsh cell disruption, there relative advantages and which tissue types work best with the particular technique.
Gentle disruption is used for softer tissues, such as tissue culture cells, whereas harsh or severe disruption is used for harder tissues, such as skeletal, connective and skin tissues that would require some form of mechanical disruption. All disruption methods, whether gentle or harsh, will also release enzymes from within the cell that can cause the break down of proteins of interest within the lysate. Considerations need to be made to prevent this if protein activity is important to the downstream work. Lysing samples in highly denaturing solutions or high pH minimizes enzymatic activity. Performing the disruption on ice or lower temperatures will also help minimize degradation of samples. One of the easiest methods is to include a general protease inhibitor cocktail to inhibit protease activity or if you are phosphorylated proteins then you would want to use a general phosphatase inhibitor cocktail.
Harsh Cell Disruption Techniques
Harsh disruption consists of mechanical homogenization, French press, sonication, bead homogenization and grinding.
- Mechanical homogenization includes using a hand-held device, like a Dounce homogenizer, or something like a blender to homogenize the tissue. This method is useful for non-seed plant type material or soft tissues (ie. Liver tissue.)
- French press uses shear force to homogenize the tissue. An example of this would be taking a cell suspension and forcing it through a narrow gauge syringe using a syringe barrel and plunger. This works well with bacteria, yeast, fungi, algae and mammalian cell culture.
- Sonication uses short bursts of ultrasonic waves to disrupt the tissue. This method generates a lot of heat and will have to be performed on ice to maintain the protein. This method is also effective for bacteria, yeast, fungi, algae and mammalian cell culture.
- Bead homogenization involves using glass or metal beads to apply gentle abrasion while vortexing them with the tissue or cell suspension.
- Grinding involves using a mortar and pestle to homogenize the tissue sample. The most common method is to freeze and grind the sample using liquid nitrogen. It can also be done using a non-binding abrasive. This method is effective for virtually all tissue types including seeds.
Gentle Cell Disruption Techniques
Gentle disruption consists of freeze-thaw lysis, enzymatic lysis, detergent lysis and osmotic lysis.
- Freeze-thaw lysis is pretty much as it sounds, using liquid nitrogen or a freezer to freeze the cells and then allow them to thaw. When cells are frozen the water inside the cells expands as it freezes causing the cells to burst open. This method is effective for mammalian cells.
- Enzymatic lysis consists of suspending the cells in iso-osmotic buffers containing enzymes that digest the cell wall (ie. Zymolyase for yeast cells, and lysozyme for bacterial cells.) This lysis method is often used in conjunction with another disruption technique (usually sonication) to ensure complete lysis of the sample. This technique is effective with bacteria, yeast, fungi, algae, non-seed plant material and mammalian cell culture.
- Detergent lysis involves suspending the cells in a detergent solution to solubilize the cell membrane, releasing the cell contents. This method also generally uses another lysis method, such as sonication to ensure complete lysis. This method is effective with mammalian cell culture.
- Osmotic lysis involves suspending cells in hypotonic (low salt) solution. This causes the cells to swell and eventually burst releasing the contents of the cells for further use. This method is also effective with mammalian cell culture.
This has been an overview of the various harsh and gentle cell disruption techniques available to extract proteins of interest. When first using cell disruption with a tissue that is new to you it is recommended to try at least two methods to see which is the most effective, as well as running a protein quantification on your final suspension to test for efficiency of the lysis. Running SDS-PAGE is also recommended to determine protein quality.
