Cell Lysates Background
Creative BioMart now offers the collection of lysates of different types which includes Over-expression lysate, Tissue lysate, Cell line lysate and Stem cell lysate. We offer tissue and cell lysates which can be used to evaluate antibody quality and serve as an excellent positive control for electrophoresis, western blotting, immunoprecipitation, enzymatic activity analysis, protein-protein interaction analysis and tissue specific expression identification, and over-expression lysates to provide a cost-effective option as assay standards in Western blot. Our lysates are validated and are available in a variety of normal, tumor and disease characterized tissues.
The lysis of cells is a central sample treatment step for studies of intracellular processes, for example in the ‘‘-omic” sciences, with the major challenge to achieve high lysis efficiency while maintaining sample integrity. Depending on purpose and cell type, mechanical, physical, chemical, and/or biological methods have been used for cell disruption. For mammalian cells, relatively mild procedures are often used, including acoustic, optical, electromagnetic, mechanical, and chemical.
Comparison of lysis techniques
The most widely used method for bulk assays which translates well to single cells, is the introduction of a chemical detergent which solubilizes lipids and proteins in the plasma membrane, creating pores which leads to complete lysis. Two common detergents, Triton X-100 and SDS, each exhibit different lysis capabilities, with the former typically inducing lysis in ~30s while preserving enzyme activity; whereas, the latter usually results in faster lysing (< 2s) but denatures membrane and cellular proteins. Denatured proteins are typically unfavorable as they quickly aggregate, forming an insoluble, randomly organized structure. While chemical lysis does not require specialized equipment apart from a mixing method, and various detergents exist to enable selective lysis, it is evident that the detergents can significantly impact the outcome of the experiment due to long times to lyse, leading to excessive diffusion of cellular content. Detergents also tend to denature and break up protein complexes, while also adding an additional reagent that may eventually need to be removed from the cell lysates prior to a specific assay.
Optical lysis techniques also exist where a pulsed (~ns) laser microbeam generates a shockwave in the vicinity of the cell, followed by the formation of a cavitation bubble which, upon expansion or collapsing, ruptures the plasma membrane. The lysing speed in this method is dependent on the position of the focal point of the laser pulse and can have a range of 1–400µs, which allows cellular content to be released quickly instead of over a lengthy period, making this method ideal for studying highly dynamic cellular processes. While there is no literature available on the use of this method to selectively lyse a membrane, when leaving other organelles intact, it is theoretically possible by adjusting the pulse duration of the beam and numerical aperture of the objective used. By employing femtosecond pulses at a high repetition rate, instead of single nanosecond pulses, the energy deposited to the cell can be significantly reduced, enabling selective membrane lysis.
The mechanical lysis subjects a cell to a physical force, such as compression within a confined region, where the mechanical stress results in membrane rupture, or to sharp, physical structures which inhibit a cell’s path and punctures the membrane in a uncontrolled manner. The compression method, while capable of producing fast (~ms), complete lysis, results in a nonuniform diffusion of cellular content, and the possibility of cellular debris adhering to parts of the compression region. Similarly, mechanical lysis against physical structures typically results in cellular debris sticking to the physical structures and poor diffusion due to incomplete membrane rupture. Additionally, neither of these mechanisms lends itself to selective lysis.
Acoustical lysis by sonication utilizes ultrasonic waves to shear a cell by generating cavitation in high pressure areas. The major limitation of this method is the long time required for complete lysis (3–50s), which can result in thermal damage to the cell over extended times and excessive diffusion of cell contents. Furthermore, localization of the ultrasonic wave to lyse a single cell poses an engineering challenge and selective lysis of a cell would require significant refinement of current techniques.
Based on the limitations of other lysis techniques, and their incompatibility with other aspects of the microfluidics program, electrical lysis was chosen as the best possible technique for this project as there is strong theoretical evidence supporting selective lysis of the plasma membrane in a controlled manner due to the cosθ dependency, while other organelles remain unharmed. Additionally, electrical lysis can typically occur within 21 milliseconds after the application of a pulse and with adequate confinement, diffusion of cell contents can be made relatively uniform, while denaturation of proteins is usually not evident.