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Isolation of Proteins From Spinach Leaves There are thousands of enzymes that have been characterized in the past century Enzymes are the proteins within cells that catalyze most of the reactions necessary for cellular viability In order to study enzymes scientists must first isolate them from other cellular components Thus the techniques employed for the purification of proteins from cells are essential and broadly applicable to protein enzyme research There are numerous methods to isolate proteins One method known as Ammonium Sulfate Precipitation precipitates proteins out of solution by adding ammonium sulfate The incremental addition of ammonium sulfate changes the solubility of proteins in solution Since ammonium sulfate has a higher affinity for water than most proteins do water tends to interact with the salt instead of the proteins This results in less interactions of proteins with water as salt is added to a protein solution In a high enough salt concentration the salt ions will interact with and displace all of the water molecules interacting with the proteins When the protein is no longer participating in hydrogen bonding with water it then precipitates This precipitate can then be collected as a pellet following centrifugation of the sample The resulting pellet theoretically contains all of the precipitated protein in the cell However this pellet usually contains more than one protein In most cases further purification steps are needed in order to completely purify the protein you wish to study In addition to ammonium sulfate precipitation you will also perform an ion exchange chromatography SDS PAGE and Western blot analysis After proteins are separated by solubility in the ammonium sulfate precipitation ion exchange chromatography will further separate the proteins based upon their net negative charge Proteins that are negatively charged will adhere to the positively charged beads that make up the resin of the ion exchange column Figure 1 The proteins disassociate from the beads in the presence of NaCl as the chloride anion displaces the proteins interactions with the beads Lower NaCl wil disassociate weakly bound proteins from the column whereas high NaCl is required to disassocial proteins that have a high affinity for the column During ion exchange chromatography the elue is collected as fractions and assayed for protein concentration by determining the absorbance 280 nm The fractions with high protein concentration are retained

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