RNAPpurification. When I was a graduate student, the first protein that I purified was RNA polymerase (RNAP) from E. colicells. RNAP has a net negative charge at physiological pH (7.4), although it binds and transcribes negatively charged DNA. After breaking open cells, I precipated RNAP and other proteins in the cell extract by adding solid (NH4)2SO4to a final concentration of 4 M. I spun down the protein precipitate in a centrifuge and dissolved it in a small amount of water buffered with 10 mM Tris pH 7.4.
A)Before performing ion-exchange chromatography, I dialyzed the protein solution against 10 mM Tris pH 7.4. Why was this necessary? [50words or less]
B)Which type of ion-exchange resin will bind RNAP at pH 7.4?
C)I eluted RNAP from this resin by applying an increasing concentration of KCl to the top of the column. Why did KCl elute RNAP?
D)RNAP was further purified by affinity chromatography. What is an excellent ligand to attach to an inert resin, which will bind RNAP with high affinity and specificity? [Think about a specific DNA sequence]
E)Finally, I obtained homogeneous RNAP after resolving it by gel-filtration HPLC. RNAP eluted from this column asa 450-kDa protein. However, when I analyzed the pure RNAP by SDS-PAGE, I observed four polypeptides of 37, 70, 151, and 155 kDa. Explain these contrasting results
RNAPpurification. When I was a graduate student, the first protein that I purified was RNA polymerase (RNAP) from E. colicells. RNAP has a net negative charge at physiological pH (7.4), although it binds and transcribes negatively charged DNA. After breaking open cells, I precipated RNAP and other proteins in the cell extract by adding solid (NH4)2SO4to a final concentration of 4 M. I spun down the protein precipitate in a centrifuge and dissolved it in a small amount of water buffered with 10 mM Tris pH 7.4.
A)Before performing ion-exchange chromatography, I dialyzed the protein solution against 10 mM Tris pH 7.4. Why was this necessary? [50words or less]
B)Which type of ion-exchange resin will bind RNAP at pH 7.4?
C)I eluted RNAP from this resin by applying an increasing concentration of KCl to the top of the column. Why did KCl elute RNAP?
D)RNAP was further purified by affinity chromatography. What is an excellent ligand to attach to an inert resin, which will bind RNAP with high affinity and specificity? [Think about a specific DNA sequence]
E)Finally, I obtained homogeneous RNAP after resolving it by gel-filtration HPLC. RNAP eluted from this column asa 450-kDa protein. However, when I analyzed the pure RNAP by SDS-PAGE, I observed four polypeptides of 37, 70, 151, and 155 kDa. Explain these contrasting results
Related textbook solutions
Basic Chemistry
Principles of Chemistry Molecular Approach
Chemistry: Structure and Properties
Principles of Chemistry Molecular Approach
Chemistry: A Molecular Approach
Chemistry: A Molecular Approach
Principles of Chemistry: A Molecular Approach
