Question

Please answer questions 2,3,4,6,7, and in regards to Hydrophobic interaction chromatography.

1. Explain how placing the colony in nutrient broth relates to your goal of purifying GFP. 2. What is the function of the following? a. Lysozyme- b. Freezer c. Centrifugation 3. You picked one white and one fluorescent colony, yet both broth cultures were green. Why? 4. Why did the outer membrane of the cell rupture when the cells were frozen? (HINT: think about unopened, frozen soft drink cans) 5. In Step IV, what color was the pellet? What color was the sup? What did this tell you? 6. Describe hydrophobic interaction chromatography 7. How are column retention and elution times related? 8. Knowing what you now know about HIC, rank the four buffers used in this procedure in terms of salt content from highest to lowest.

Answer 1

2. Lysozyme function- it is an enzyme which cleaves the peptidoglycan layer present in the gram positive bacterial cell wall. Also, it cleaves the glycosidic bonds that connects N-acetylmuramic acid with N-acetylglucosamine. This enzyme is found in number of human secretions such as saliva, tears, human milk as to protect against the pathogen.

Freezer- This has sub-zero temperature going upto -200C. This is used to freeze food or biological products as to protect them against being spoiled.

Centrifuge- this is used to spin some solutions and precipitate products out of them at the end of the tube under the influence of centrifugal force.

6. Hydrophobic interaction chromatography is used to separate and purify proteins using high salt solutions. Proteins are made up of different types of amino acids- Hydrophilic, neutral and hydrophobic amino acids. These amino acids are found in a particular orientation with mostly hydrophobic amino acids being present in the inner most areas of properly folded protein. Hydrophilic amino acids are found on the outer areas of proteins.

HIC is based on salting out principle of proteins. When proteins are kept in aqueous solutions, the water molecules interact with the hydrophilic areas of the protein. Now when salt is added in this solution at a very high concentration, water molecules starts to interact with the salt ions. During this water-salt interactions, protein molecules present in the solution starts to interact more effectively with each other and precipitate out. HIC consists of a solid support which has hydrophobic molecules on it. These molecules on solid support interact with hydrophobic areas of the precipitated proteins from the high salt solutions and purify them.

HIC can bind proteins to different extents depending on protein to be precipitated, salt concentration, pH, temperature etc.