Question

6a. You have found a new protein of interest: this protein has a transmembrane domain (as predicted by hydropathy analysis). You want to confirm this and determine which portion of the protein is outside the cell and which portion is inside the cell. You begin with a protease protection assay, the results are below. Does this protein most likely have a transmembrane domain? Is the larger portion inside the cell or outside the cell? Is there anything wrong with the results seen in the 3rd lane? Does this change your answer? (8) + Protease + Detergent 65. Next you want to use a complementary assay to ask the same question, is this protein a transmembrane protein, so you treat the cells with detergent and then perform a sup/pellet assay. This means you centrifuge the cells, anything found in the supernatant is soluble, anything found in the pellet is still associated with the membrane. Below are the treatments and your results. S=supernatant, P=pellet. Is this protein a transmembrane protein? How do you know? (6) 0% S 0% P 2% S 2% P 10% 10% Detergent S P

Answer 1

• The detergents are used to lyse the cell which means that the to release the soluble proteins and the components inside the cell so thet through centrifuge we can extract the DNA from the cell.
• 6a) In this the proteins are likely to be inside of the cell membrane because the integral proteins will be protected within the lipidbi layer the protein cannot be lysed without the use of harsh detergents.
• 6b) In this the detergents has been used to lyse the cell. So the proteins lipid layers are ruptured and the at the time of centrifuge the protein moves outside of the cell. And those components that soluble in water and detergents are dissolved and called as supernanant.
• So this protein is a trans membrane protein. Since during the use of detergents the pallet is fully dissolved.