Question

Sample is homogenized bamburger.

Add ammonium sulfate

Questions of lab for isolation of crude myoglobin

a. What cellular materials does the pellet contain after the first centrifugation? [1.0]

b. When adding ammonium sulfate the first time you are bringing the solution to 60% ammonium sulfate saturation. At this point, what do you predict is in the supernatant? Why? What do you predict is in the pellet, and why? [2.0]

c. What is contained in the pellet and supernatant after salting out (up to 100% saturation)? Why? [2.0]

d. What is the purpose of dialysis and how is this important for the success of the next step in the purification process, ion-exchange chromatography? [3.0]

Answer 1

1. Whole broken cells, nuclei and cytoskeleton

2. Proteins which are hydrophobic in nature will be precipitated out and can be seen in the pellet. the hydrophilic protein will be in the supernatant due to salting in

3. All the proteins will be salted out. So, it will be seen in the pellet. Due to ionic repulsion it will occur. The other cellular parts will be in the supernatant.

4. The ammonium sulfate is removed by dialysis. If those are present during ion exchange chromatography, then the NH4+ and SO42- ions will interfere during the purification process