You treat some cells with a proteolytic enzyme that is too largeto penetrate the cell membrane (Set 1). Another group of cells ismade permeable before treatment with the enzyme (Set 2). A thirdset of cells was not treated with the enzyme at all (controls).Proteins are then extracted from the three different sets of cellsand applied to an SDS-PAGE gel. Protein W migrates to the samedistance on a gel of proteins from Set 1 and Set 2; Protein Wmigrates a shorter distance on gels of proteins extracted from thecontrol group than on gels of proteins extracted from Set 1 and Set2 treated cells. Protein X migrates to the same distance on a gelof proteins from control cells and the gels of the proteins fromSet 1 and Set 2. Protein Y migrates a longer distance whenextracted from Set 1 cells than does protein Y in the controls;Protein Y moves an even larger distance in the gel of the extractsfrom Set 2. Protein Z migrates the same distance on gels ofproteins from the controls and the proteins extracted from Set 1,but it migrates a longer distance in extracts from Set 2 cells.
A. Draw a figure to represent what this question is asking.Label where each protein should be located in the membrane.
B. Which protein is exposed only on the exterior of thecell?
C. Which protein in the question above is likely to be burriedwithin the membrane?
You treat some cells with a proteolytic enzyme that is too largeto penetrate the cell membrane (Set 1). Another group of cells ismade permeable before treatment with the enzyme (Set 2). A thirdset of cells was not treated with the enzyme at all (controls).Proteins are then extracted from the three different sets of cellsand applied to an SDS-PAGE gel. Protein W migrates to the samedistance on a gel of proteins from Set 1 and Set 2; Protein Wmigrates a shorter distance on gels of proteins extracted from thecontrol group than on gels of proteins extracted from Set 1 and Set2 treated cells. Protein X migrates to the same distance on a gelof proteins from control cells and the gels of the proteins fromSet 1 and Set 2. Protein Y migrates a longer distance whenextracted from Set 1 cells than does protein Y in the controls;Protein Y moves an even larger distance in the gel of the extractsfrom Set 2. Protein Z migrates the same distance on gels ofproteins from the controls and the proteins extracted from Set 1,but it migrates a longer distance in extracts from Set 2 cells.
A. Draw a figure to represent what this question is asking.Label where each protein should be located in the membrane.
B. Which protein is exposed only on the exterior of thecell?
C. Which protein in the question above is likely to be burriedwithin the membrane?
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You further investigate the regulatory proteins that bind to Regions 1, 2 and 3 using gel shift assays. A gel shift assay uses labeled DNA as a target for regulatory protein binding.
A labeled DNA fragment that fails to bind a protein will migrate quickly on an electrophoretic gel.
A labeled DNA fragment that does bind a protein will migrate more slowly because the DNA + protein complex is larger than the DNA alone. Thus, the label associated with the DNA will âshiftâ to a higher location on the gel, as shown in this diagram.
To investigate regulatory protein binding to Regions 1, 2, and 3, you will use labeled DNA fragments with some of the same point mutations as in Part B, incubate them with proteins extracted from heart or lung tissue, and conduct a gel shift assay. How would gel shift assay results be affected by these mutations?
Select the two correct answers.
| 1. Region 2 binds protein from lung tissue. If Region 2 is mutated, then the DNA will not shift on the gel when incubated with protein from lung tissue. |
| 2. Regions 1 and 3 bind protein from heart tissue. If both Regions 1 and 3 are mutated, then the DNA will not shift on the gel when incubated with protein from heart tissue. |
| 3. Region 2 binds protein from heart tissue. If Region 2 is mutated, then the DNA will not shift on the gel when incubated with protein from heart tissue. |
| 4. Regions 1 and 3 bind protein from lung tissue. If both Regions 1 and 3 are mutated, then the DNA will not shift on the gel when incubated with protein from lung tissue. |
