polymerase 4. RNAP purification. When I was a graduate student, the first protei
ID: 548084 • Letter: P
Question
polymerase 4. RNAP purification. When I was a graduate student, the first protein that I purified was RNA (RNAP) from E coli cells. RNAP has transcribes negatively charged DNA. After breaking open cells, I precipated RNAP and other proteins in the cell a net negative charge at physiological plH (7.4), although it binds and extract by adding solid (NH) SO, to 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? [50 words or less B) Which type of ion-exchange resin will bind RNAP at pH 7.47 C) I eluted RNAP from this resin by applying an increasing concentration of KCI to the top of the column. Why did KCI elute RNAP? [50 words or less 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. 10 words or less] E) Finally. I obtained homogeneous RNAP after resolving it by gel-filtration HPLC. RNAP eluted from this column as a 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. 150 words or less.Explanation / Answer
A) It's to remove DNA and RNA of cell lysate.
B) Positively charged ion-exchange resin is used since is negatively charged at pH 7.4. This allows binding of RNAP to resin.
C) Increasing gradient of KCl allows elution of exchange of RNAP by negatively charged chloride ions.
D) If one knows sequence of RNAP a complimentary DNA primer will allow bindig of RNAP with high affinity and specificity.
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