Passage III One way to use the principle of density to isolate macromolecules from cell extracts is by separating out the various parts of a cellular extract using a density gradient 4 A Experiment 1 To make the density gradient 6 mL aliquots for each step a layer of liquid that has a different density than the other layers of the density gradient were made by mixing together pure water with different amounts of 60 wt vol iodixanol solution The density of the 60 iodixanol solution is known 1 32 g mL See Table 1 for measurements of the various steps of the density gradient Step 1 2 3 4 5 Mass of water g 0 00 0 91 1 69 2 48 3 00 Table 1 4 4 3 Set Three Mass of iodixanol Total mass Density g mL g solution g 8 10 6 87 5 81 4 73 4 05 8 10 7 78 7 50 7 21 7 05 1 35 1 30 1 25 1 20 1 18 Deriment 2 To make steps in the density gradient that are lower sity than Step 5 a Falcon tube was tared on an electronic nce Water and acetonitrile were added and weighed in Falcon tube until the total volume in the Falcon tube was Several more steps of the density gradient were made dding different masses of water and acetonitrile the urements for which are noted in Table 2 Step 6 7 8 9 10 Condition 6 00 5 00 4 00 3 00 2 00 virus caspase 1 virus caspase 5 Mass of Mass of Total mass Density water g acetonitrile g g g mL virus only virus trypsin virus proteinase K virus MMP 9 Table 2 11 0 00 0 78 1 56 2 35 3 13 Experiment 3 To determine the various densities of HPV virus fragments that are processed by the cell and by extracellular proteases 293TT cells were infected with HPV and then harvested after 18 hours To some of the cellular extracts proteases such as trypsin proteinase K MMP 9 caspase 1 and caspase 5 were added and incubated with the extracts prior to loading them onto the density gradient To locate where the HPV virus was on the gradient it was tagged with GFP prior to infection the virus will show up as a green band in one or more of the steps of the gradient The locations of the HPV virus on the gradient after the various conditions are listed in Table 3 means that GFP was detected in that step of the gradient and means GFP was not detected in that step of the density gradient Table 3 11 1 Step of the density gradient 1 2 3 4 5 6 7 8 9 10 1111 6 00 5 78 5 56 5 35 5 13 TING 1 00 0 96 0 93 0 89 111 11 11 II 1 1 III 0 85 E of Hypotheses Part 2 4 A A A A A A A A A 4 11 The data from Table 3 was interpreted to mean that MMP 9 does not affect the location of the HPV virus on the gradient Is that interpretation correct A No the GFP signal from the virus MMP 9 condition is vastly different from that of virus alone B Yes the GFP signals from the virus alone and virus are same MMP 9 C No the GFP signal from the virus MMP 9 condition was seen in the steps of the gradient with the least density D Yes the GFP signal from virus MMP 9 was similar to that of the virus caspase 1 condition 12 A student predicted that if a solid with a known density of 2 33 g mL were mixed with the solution from Step 5 of Experiment 1 the solid would float to the top Based on the information in Table 1 is the student s prediction correct F Yes because the mass of the solid 2 33 g is less than mass of the Step 5 iodixanol solution 7 05 g G Yes because the density of the solid is less than the of the solutions in the table density of H No because the solid would dissolve and create a homogenous solution J No because the density of the solid is greater than the density of the solution from Step 5 13 Do the results of Experiment 2 indicate that the density of acetonitrile is less than the density of water 14 A student stated that the location of the HPV virus on the gradient is affected by each protease in different ways Do the results of Experiment 3 verify this statement A Yes the table indicates that only 3 13 g of acetonitrile can be added to the solution while a maximum of 6 g of water can be added to the same solution B Yes as more acetonitrile and less water is added to the same volume of solution the density goes down indicating acetonitrile has a lower density C No the density of acetonitrile solutions goes down the more water is added indicating that water has a lower density D No the mass of water added in each step goes down while the density goes up showing that acetonitrile has a higher density than water F Yes because the results of Table 3 show that the location of the virus was detected in different steps for each protease G Yes because the results of Table 3 show that the location of the virus was detected in the same steps for each protease H No because the results of Table 3 show that the location of the virus was detected in different steps for each protease J No because the results of Table 3 show that the location of the virus was detected in the same steps for each protease 15 Is the statement caspase 5 is denser than caspase 1 supported by the information in Table 3 A Yes because caspase 5 reacts with the HPV virus at densities of 5 and 6 while caspase 1 reacts with the HPV virus at densities of 4 and 5 B Yes because out of all of the proteases listed in the table caspase 5 is the densest as shown by its placement in Table 3 and its reaction with the HPV virus C No because caspase 5 reacts with the HPV viru at later steps of the gradient indicating that it has lower density than caspase 1 D No because the table does not compare the densit of the various proteases but instead shows h the relative densities of HPV virus fragments produced by the different proteases END OF SET STOPI DO NOT GO ON TO THE NE