Alkaline phosphatase, or AP, is an enzyme responsible for the dephosphorylation, or removal of phosphate groups, from various types of molecules such as proteins and nucleotides. It has a molecular weight of 140 to 160 kilo-Daltons and, as the name suggests, alkaline phosphatase functions in an environment with pH values ​​between 7.5 and 9.5, which is an alkaline or basic environment. . And according to Sigma-Aldrich, alkaline phosphatase can be used to "dephosphorylate the 5' ends of DNA or RNA to prevent self-ligation" (sigmaaldrich.com). Alkaline phosphatase in Gram-negative bacteria can be found in the periplasmic space, which lies outside the cell membrane. For this reason, it is subjected to various types of environmental activities, making alkaline phosphatase more resistant to denaturation, inactivation and degradation, while giving the PA a higher activity rate . Every cell has a plasma membrane, which is a protein. lipid bilayer. It forms a barrier that separates cellular contents from the external environment. Amphipathic lipids constitute the plasma membrane, that is to say they have hydrophilic and hydrophobic parts. The hydrophilic heads and hydrophobic tails form a “sheet” containing lipids whose hydrophobic tails face each other. In order to get something from inside the cell, or in this case alkaline phosphatase, one could use the process of cell lysis. Cell lysis is a process that breaks down or destroys a cell wall due to an external force. Cell lysis can occur naturally or artificially. Natural lysis can occur as a result of viral infections, while artificial lysis can occur by adding a substance that can destroy a cell wall. One such substance that can cause artificial cell lysis is lysozyme. Lysozyme, or N-acetyl...... middle of paper ......tions collected, a spot test can be used. The spot test is a qualitative determination to see if a molecule exists in solution. For this one-time test, para-nitrophenylphosphate, PNPP, in solution was used to determine the fractions of the IEC containing or not containing AP. PNPP was used specifically because when PNPP comes into contact with PA under basic conditions, a phosphate from the PNPP is cleaved and becomes para-nitrophenol, PNP. PNP turns yellow under basic conditions. If AP was in a fragment, the yellow hue shown would indicate this. The experiment was performed using mutated E. coli, cell lysis, two centrifugations, two dialysis processes, thermal denaturation, release via ammonium sulfate, anion exchange and spot chromatography. essay. By following these procedures one should be able to obtain plenty of purified alkaline phosphatase and one should see a yellow tint when spot testing..