text: In every experiment, it is important to determine the relative concentration of the protein in question, ammonium persulfate, and Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate that will be used in the protein cross-linking experiment. Previous experiments have shown that for amyloid β-protein (Aβ), the peptide assumed to cause toxicity in Alzheimer's disease, the ratio of protein: Ru(Bpy): APS is 1:2:40. The ratio of Ru(Bpy) and APS is suggested to be kept at this ratio, but the appropriate concentration of a given protein can vary. For many proteins that PICUP has not yet been used for, finding the appropriate concentrations can be done through trial and error. Generally, protein concentrations would fall in between 10 and 50μM, dissolved in the corresponding buffer, most likely sodium phosphate if testing for conditions at physiological pH. However, some studies of pure protein suggest that the protein to Ru(Bpy) ratio should be kept at 1:2 as well. This level arises from the fact that the lower amount of Ru(Bpy) can lead to the protein sample appearing to have more than the actual number of higher order oligomers, and a greater amount of Ru(Bpy) can allow for artificial cross-linking byproducts.
  The general method for PICUP is as follows:
  # The appropriate amount of the protein is pipetted into the polymerase chain reaction tube (PCR). 
  # Ammonium persulfate (APS) and Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate are added to the sample and mixed briefly. 
  # The PCR tube is placed in a glass vial to hold it still inside the bellows of the camera that would irradiate the mixture. Upon closing the lid that covers the camera, pressing down the camera shutter illuminates the tube for however long you program the camera to irradiate for. Using the same camera for every replicate of the experiment ensures that the irradiation time and the distance from the light sourced is controlled for every PICUP experiment.
  # After the sample in the PCR tube is irradiated, a calculated amount of 1M Dithiothreitol (DTT) is immediately added to the mixture to quench the reaction. If the reaction is not quenched, the oligomers will continually aggregate and lower order oligomers will not be present in the mixture. In addition, if SDS-PAGE is to be used to analyze the oligomer distribution of the proteins after PICUP, DTT would also act as a denaturing agent to the proteins before gel electrophoresis.