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The ideal salt for protein precipitation is most effective for a particular amino acid composition, inexpensive, non-buffering, and non-polluting. The most commonly used salt is ammonium sulfate . There is a low variation in salting out over temperatures 0 °C to 30 °C.
As different proteins have different compositions of amino acids, different protein molecules precipitate at different concentrations of salt solution. [citation needed] Unwanted proteins can be removed from a protein solution mixture by salting out as long as the solubility of the protein in various concentrations of salt solution is known.
Ammonium sulfate precipitation is a useful technique as an initial step in protein purification because it enables quick, bulk precipitation of cellular proteins. [4] It is also often employed during the later stages of purification to concentrate protein from dilute solution following procedures such as gel filtration .
In an aqueous solution, precipitation is the "sedimentation of a solid material (a precipitate) from a liquid solution". [1] [2] The solid formed is called the precipitate. [3] In case of an inorganic chemical reaction leading to precipitation, the chemical reagent causing the solid to form is called the precipitant. [4]
Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules and often by disrupting the hydrophobic interactions that give many proteins their tertiary structure. The precipitation and aggregation of proteins is a very different process from the crosslinking that occurs with aldehyde fixatives.
The Bradford protein assay (also known as the Coomassie protein assay) was developed by Marion M. Bradford in 1976. [1] It is a quick and accurate [2] spectroscopic analytical procedure used to measure the concentration of protein in a solution. The reaction is dependent on the amino acid composition of the measured proteins.
RIPA buffer is a commonly used lysis buffer for immunoprecipitation and general protein extraction from cells and tissues. The buffer can be stored without vanadate at 4 °C for up to 1 year. [ 10 ] RIPA buffer releases proteins from cells as well as disrupts most weak interactions between proteins.
Immunoprecipitation of intact protein complexes (i.e. antigen along with any proteins or ligands that are bound to it) is known as co-immunoprecipitation (Co-IP). Co-IP works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins.