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3) You are studying an enzyme of interest and decide to purify the enzyme using affinity chromatography. You utilize what is called a FLAG-tag: a short peptide sequence of eight amino acids that binds a highly specific antibody, anti-FLAG. You express a fusion protein of your enzyme and the FLAG sequence in cell culture and collect the cell lysate. You use a column filled with agarose beads conjugated to anti-FLAG to purify the FLAG-tagged enzyme from cell lysate. You apply the lysate to the column with binding buffer and allow the binding buffer to run through the column. Then, you add elution buffer containing free FLAG peptide to release the FLAG-tagged protein from the column. You collect fractions throughout this process. You plot the absorbance at 280nm and the enzymatic activity of your fractions, as shown: absorbance and enzymatic activity of fractions binding phase elution phase Fractions Why do you observe two peaks of absorbance at 280nm and only one enzymatic activity peak? Select the best option. a) The FLAG-tagged enzyme that flows out during the binding step is not active. b) The left absorbance peak corresponds to other proteins in the lysate. c) The enzymatic activity is from free FLAG peptides in the elution buffer. d) The enzyme is not active until cleaved from the FLAG-tag in the elution step. Enzyme activity Absorbance 280 nm

3) You are studying an enzyme of interest and decide to purify the enzyme using affinity chromatography. You utilize what is called a FLAG-tag: a short peptide sequence of eight amino acids that binds a highly specific antibody, anti-FLAG. You express a fusion protein of your enzyme and the FLAG sequence in cell culture and collect the cell lysate. You use a column filled with agarose beads conjugated to anti-FLAG to purify the FLAG-tagged enzyme from cell lysate. You apply the lysate to the column with binding buffer and allow the binding buffer to run through the column. Then, you add elution buffer containing free FLAG peptide to release the FLAG-tagged protein from the column. You collect fractions throughout this process. You plot the absorbance at 280nm and the enzymatic activity of your fractions, as shown: absorbance and enzymatic activity of fractions binding phase elution phase Fractions Why do you observe two peaks of absorbance at 280nm and only one enzymatic activity peak? Select the best option. a) The FLAG-tagged enzyme that flows out during the binding step is not active. b) The left absorbance peak corresponds to other proteins in the lysate. c) The enzymatic activity is from free FLAG peptides in the elution buffer. d) The enzyme is not active until cleaved from the FLAG-tag in the elution step. Enzyme activity Absorbance 280 nm

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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3) You are studying an enzyme of interest and decide to purify the enzyme using affinity chromatography. You utilize what is called a FLAG-tag: a short peptide sequence of eight amino acids that binds a highly specific antibody, anti-FLAG. You express a fusion protein of your enzyme and the FLAG sequence in cell culture and collect the cell lysate. You use a column filled with agarose beads conjugated to anti-FLAG to purify the FLAG-tagged enzyme from cell lysate. You apply the lysate to the column with binding buffer and allow the binding buffer to run through the column. Then, you add elution buffer containing free FLAG peptide to release the FLAG-tagged protein from the column. You collect fractions throughout this process. You plot the absorbance at 280nm and the enzymatic activity of your fractions, as shown: absorbance and enzymatic activity of fractions binding phase elution phase Fractions Why do you observe two peaks of absorbance at 280nm and only one enzymatic activity peak? Select the best option. a) The FLAG-tagged enzyme that flows out during the binding step is not active. b) The left absorbance peak corresponds to other proteins in the lysate. c) The enzymatic activity is from free FLAG peptides in the elution buffer. d) The enzyme is not active until cleaved from the FLAG-tag in the elution step. Enzyme activity Absorbance 280 nm
Transcribed Image Text:3) You are studying an enzyme of interest and decide to purify the enzyme using affinity chromatography. You utilize what is called a FLAG-tag: a short peptide sequence of eight amino acids that binds a highly specific antibody, anti-FLAG. You express a fusion protein of your enzyme and the FLAG sequence in cell culture and collect the cell lysate. You use a column filled with agarose beads conjugated to anti-FLAG to purify the FLAG-tagged enzyme from cell lysate. You apply the lysate to the column with binding buffer and allow the binding buffer to run through the column. Then, you add elution buffer containing free FLAG peptide to release the FLAG-tagged protein from the column. You collect fractions throughout this process. You plot the absorbance at 280nm and the enzymatic activity of your fractions, as shown: absorbance and enzymatic activity of fractions binding phase elution phase Fractions Why do you observe two peaks of absorbance at 280nm and only one enzymatic activity peak? Select the best option. a) The FLAG-tagged enzyme that flows out during the binding step is not active. b) The left absorbance peak corresponds to other proteins in the lysate. c) The enzymatic activity is from free FLAG peptides in the elution buffer. d) The enzyme is not active until cleaved from the FLAG-tag in the elution step. Enzyme activity Absorbance 280 nm
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