Problem 9
REFLECT AND APPLY You are purifying a protein for the first time. You have solubilized it with homogenization in a blender followed by differential centrifugation. You wish to try ammonium sulfate precipitation as the next step. Knowing nothing beforehand about the amount of ammonium sulfate to add, design an experiment to find the proper concentration (\% saturation) of ammonium sulfate to use.
Problem 11
RECALL What is the basis for the separation of proteins by the following techniques? (a) gel-filtration chromatography (b) affinity chromatography (c) ion-exchange chromatography (d) reverse phase HPLC
Problem 13
RECALL What are two ways that a compound can be eluted from an affinity column? What could be the advantages or disadvantages of each?
Problem 18
RECALL How can gel-filtration chromatography be used to arrive at an estimate of the molecular weight of a protein?
Problem 19
REFLECT AND APPLY Sephadex \(G-75\) has an exclusion limit of 80,000 molecular weight for globular proteins. If you tried to use this column material to separate alcohol dehydrogenase (MW 150,000 ) from \(\beta\) -amylase \((\mathrm{MW} 200,000),\) what would happen?
Problem 22
RECALL How does HPLC differ from ion-exchange chromatography?
Problem 25
REFLECT AND APPLY What could be an advantage of using an anion exchange column based on a quaternary amine [i.e. resin-N \(\left.^{+}\left(\mathrm{CH}_{2} \mathrm{CH}_{3}\right)_{3}\right]\) as opposed to a tertiary amine [resin-NH \(^{+}\) \(\left.\left(\mathrm{CH}_{2} \mathrm{CH}_{3}\right)_{2}\right] ?\)
Problem 26
REFLECT AND APPLY You wish to separate and purify enzyme A from contaminating enzymes \(\mathrm{B}\) and \(\mathrm{C}\). Enzyme \(\mathrm{A}\) is found in the matrix of the mitochondria. Enzyme \(B\) is embedded in the mitochondrial membrane, and enzyme \(C\) is found in the peroxisome. Enzymes A and B have molecular weights of 60,000 Da. Enzyme C has a molecular weight of 100,000 Da. Enzyme A has a pI of 6.5 Enzymes B and C have pI values of \(7.5 .\) Design an experiment to separate enzyme A from the other two enzymes.
Problem 27
REFLECT AND APPLY An amino acid mixture consisting of lysine, leucine, and glutamic acid is to be separated by ion-exchange chromatography, using a cation-exchange resin at \(\mathrm{pH} 3.5\), with the eluting buffer at the same pH. Which of these amino acids will be eluted from the column first? Will any other treatment be needed to elute one of these amino acids from the column?
Problem 28
REFLECT AND APPLY An amino acid mixture consisting of phenylalanine, glycine, and glutamic acid is to be separated by HPLC. The stationary phase is aqueous and the mobile phase is a solvent less polar than water. Which of these amino acids will move the fastest? Which one will move the slowest?
