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A cell-free system is an in vitro tool widely used to study biological reactions that happen within cells apart from a full cell system, thus reducing the complex interactions typically found when working in a whole cell. [1]
Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. The in vitro protein synthesis environment is not constrained by a cell wall or homeostasis conditions necessary to maintain cell viability. [ 1 ]
Lysis (/ ˈ l aɪ s ɪ s / LY-sis; from Greek λῠ́σῐς lýsis 'loosening') is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic (that is, "lytic" / ˈ l ɪ t ɪ k / LIT-ik) mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a lysate.
The procedure begins by making a cell lysate of the cells of interest. This lysate contains polysomes, monosomes (composed of one ribosome residing on an mRNA), the small (40S in eukaryotes) and large (60S in eukaryotes) ribosomal subunits, "free" mRNA and a host of other soluble cellular components.
Limulus amebocyte lysate (LAL) is an aqueous extract of motile blood cells from the Atlantic horseshoe crab Limulus polyphemus. LAL reacts with bacterial endotoxins such as lipopolysaccharides (LPS), which are components of the bacterial capsule , the outermost membrane of cell envelope of gram-negative bacteria .
In order to decipher this biological mystery, Nirenberg and Matthaei needed a cell-free system that would build amino acids into proteins. Following the work of Alfred Tissieres and after a few failed attempts, they created a stable system by rupturing E. coli bacteria cells and releasing the contents of the cytoplasm. [ 7 ]
FBS-free cell culture media, e.g. with platelet lysate or chemically defined/ animal component free, are used for cell therapy or regenerative medicine. They are commercially available in GMP ( good manufacturing practice )-quality which is generally basis for regulatory approval.
In most cases, preclearing the lysate at the start of each immunoprecipitation experiment (see step 2 in the "protocol" section below) [6] is a way to remove potentially reactive components from the cell lysate prior to the immunoprecipitation to prevent the non-specific binding of these components to the IP beads or antibody.