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Cell Lysis Method Protocol

Information on the cell lysis technique here.

Cell Lysis Methods Table of Contents

What is Cell Lysis?

Cell lysis or cellular disruption is a cell biology method for the release of biological molecules including organelles, proteins, DNA, RNA and lipids from inside a cell.

cell lysis

Background on Cell Lysis?

For most cells, mild osmosis is usually enough to lyze the cells. This is done by lowering the ionic strength of the surrounding solution, causing the cells to swell and burst releasing their contents.

Mild surfactans are often used in addition to mechanical lysis methods.

Mechanical or Physical Lysis Methods include:

  • mechanical agitation
  • pressure
  • sonication
  • nitrogen bomb or nitrogen burst lysis method
  • ultrasound with small probes

These steps are conducted to ensure complete dissociate of the cell and its cellular components including breakdown of organellar and nuclear membranes.

Due to the high costs of growing cells, usually only a small amount of cellular material is available. It is thus necessary to be efficient when lysing cells to ensure the maximum of usable material is obtained. Often, several methods are used in combination to ensure almost complete lysis.

Applications of Cell Lysis

Cell lysis is vital for the extraction of DNA, RNA and Proteins from cells. Therefore, it is needed in most cell biology techniques and even molecular biology techniques.

Limitations of Cell Lysis

Cell lysis is limited in its usage as it requires the cell membrane to be pierced and cell contents to be released. These lysed cells die and cannot be cultured or grown again.

Factors to Consider for Cell Lysis

There are many factors to consider which cell lysis method to use or how to conduct it.

Volume of Cells

The amount of cells to lyse is an important parameter in cell lysis. If you only have a very

If only a few microliters of sample are available, care must be taken to minimize loss and to avoid cross-contamination.

Disruption of cells, when hundreds or even thousands of liters of material are being processed in a production environment, presents a different challenge. Throughput, efficiency, and reproducibility are key factors.

Number of Cell Samples to Lyze

Often there are many cell samples to lyze. There are many issues to consider when doing this including cross contamination of samples, the speed of processing, and equipment cleaning after each sample is lysed.

Difficulty of Cells to Lyze

Some cells are quite difficult to lyse. As the lysis difficulty increases, a greater lysis force or higher ionic strength of buffers is required to efficiently disrupt the cells.

For more difficult lysis of cell samples such as yeast samples, there is a steep increase in the processing power, time and cost required.

For the most difficult samples to lyze, such as bacterial spores, these require mechanical forces combined with chemical or enzymatic efforts. The problem is even with multiple and harsher methods, usually one achieves limited disruption efficiencies.

Efficiency of Cell Lysis

Depending on what part of the cell, organelle or fraction you need to isolate, over-lysis may affect your desired product.

If subcellular fractionation is used, it is more important to have a delicate lysis to achieve an intact subcellular organelle components. Obviously, you will achieve a lower efficiency of lysis and thus will require more cells. To scale these cell lysis processesses, the time to disrupt the cells and the reproducibility of the method become more important factors.

Cell Lysis vs Molecule to be Isolated

It is very important to cater the lysis method to the protein you need isolated. If it is a nuclear protein, care is required to lyse the cell first and then isolate the nuclear membrane. After the nuclear membrane is isolated, then one may lyse the nuclear membrane and release the molecule of interest. This methodology decreases the contamination of molecules that are from other cellular compartments/organelles.

Furthermore, depending on the molecule care must be taken to use or not use certain lysis solutions or methods. For example, if you are trying to isolate a phospho-protein that is sensitive to phosphatases, it is not a good idea to lyse and expose the phospho-protein to thousands of proteases and phosphatase enzymes. Protecting your molecule from harsh or enzymatic conditions is important. The temperature (low temperature of lysis), and usage of inhibitory molecules (such as protease and phosphatase inhibitors) is important in these lysis cases.

 

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