Cell Lysis and Fractionation

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Creative Biostructure is pround to offer cell lysis and fractionation technical support in order to accelerate your protein research. It is a crucial part of life science research to study proteins in living organisms. Proteins are the fundamental materials which are essential for cellular structure and function, and they are the most diverse and important biologically molecules. The first step in protein analysis is cell lysis and fractionation.

Cell membranes and organelles can be disrupted by cell lysis, leading to unregulated proteolytic activity which can affect the protein yield and function. Protease and phosphatase inhibitors are frequently added in order to avoid degradation of extracted proteins and to obtain the best possible protein yield and activity after cell lysis process. Researchers have identified a large number of compounds that can be used to reduce or inactivate the protease activities and phosphatase activities by binding to them reversibly or irreversibly.

Since some detergents added in protein extraction formulations may cause inactivation of enzymes of interest or alter their long-term stability, it is essential to remove the detergents following cell lysis sometimes. Furthermore, high concentrations of detergents or salts prefer to interfere with some common research methods including protein assays, gel electrophoresis, purification, immunoprecipitation, and mass spectrometry. Dilution or dialysis is the simplest way to mitigate the interfering substances.

Methods used for cell lysis and fractionation are listed in the table below.

Lysis Method	Apparatus	Method	Sample Type	Fractions Isolated	Advantages	Disadvantages
Mechanical	Waring blender	Shearing caused by rotating blades	Mammalian tissues or cells	Total protein	Inexpensive equipment	Reproducibility may vary, denaturing may occur, noisy
	Bead beater	Disruption caused by collision with agitated beads in liquid suspension	Mammalian tissues or cells, yeast or bacterial cells, plant tissue	Total protein	Low shearing, works with wide range of cell and tissue types, good for hard, difficult tissues	Denaturing may occur due to heat generated, noisy
	Potter-Elvehjem homogenizer	Shearing is caused by a PTFE- coated pestle that is manually or mechanically driven into a conical vessel	Mammalian tissues or cells	Total protein	Hand-held device, ideal for small volumes	Reproducibility may vary, denaturing may occur
	Dounce homogenizer	Shearing caused by a round glass pestle that is manually driven into a glass tube	Mammalian tissues or cells	Total protein, mitochondria	Inexpensive equipment, ideal for small volumes	Reproducibility may vary, denaturing may occur
	French press	Shearing is caused by high pressure when sample is forced through a small hole	Bacterial cells	Total protein	Rapid, efficient	Minimum 40mL samples, very expensive, noisy, lengthy set-up and clean-up
Sonication	Sonicator	Shearing is caused by high frequency sound waves	Mammalian tissues or cells, yeast or bacterial cells	Total protein	Directly compatible with downstream applications	Reproducibility may vary, denaturing may occur, noisy
Manual Grinding	Mortar and pestle	Disruption is caused by shearing and ice crystals in membranes	Plant tissue	Total protein	Inexpensive	Reproducibility may vary
Reagent-based	None, except for tissues	Disruption of lipid membrane and/or cell wall	Mammalian, bacterial, yeast, insect, and plant cells and/or tissues	Total protein or subcellular fractions or organelles	Rapid, gentle, efficient, reproducible, high protein yield	Some components may need to be removed for downstream analysis
Freeze/Thaw	Freezer or dry ice/ethanol in container	Disruption is caused by the formation of ice crystals	Bacterial and mammalian cells	Total protein	Inexpensive	Multiple cycles may be required, reproducibility may vary

Troubleshooting:

1. How to increase lysis efficiency?
The sample type matters. Some cell types are easier for lysis than others. For example, the animal cells only contain one barrier that separate cell contents from the environment--the plasma membrane, while a rigid cell wall surrounds plasma membrane in plants and bacteria.
Detergent removal is important as well.

2. Degradation of target protein
Using a protease inhibitor;
Make sure all lysis steps were performed at 4˚C.

3. Overexpression
Some proteins can be overexpressed into insoluble and misfolded inclusion bodies. You need to adjust and optimize the expression conditions to obtain soluble protein.

4. Low yield
Optimizing the transfection procedure if the target protein was transfected into the cell line.
Make sure the recommended amount of lysis reagents was used.
Increasing the incubation time.
More vigorously mixing.

Creative Biostructure has a rich-experienced support team to offer a wide variety of technical resources in protein purification which are listed as below. We are more than happy to help you and address your research problems. Please feel free to Contact Us.