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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|
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.
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.