You are given an equimolar (0.10 mM) mixture of Ubiquitin protein that is free/pure of any other macromolecules (e.g. nucleic acids) in a pH 7.0 phosphate bufferHow can you purify Ubiquitin using Ion-Exchange chromatography (IEC)? Propose a plan using steps to purify this protein.Basic rules: Affinity chromatography (e.g. His Tag purification) is not an allowed step because these proteins are in their native state (i.e. – do not have a polyhistidine tag).Also, you are not allowed to rely solely on the color of certain proteins (e.g. cytochrome C and GFP) in your characterization/proposal.You are allowed to buffer exchange (i.e. – switch buffers) but try to keep your pH’s in a reasonable range (5.5 – 8.5) or you risk denaturing your protein.Assume proteins with the same charge (positive or negative) are not easily separated using IEC.Assume all proteins can be resolved/visualized on an SDS-PAGE gel.Ubiquitin Molecular weight (8663.02); pI (6.56); ε (M-1 cm-1) ignoring C’s (2980)Describe your first proposed step clearly in words (1-2 sentences) including the buffer/pH used.Sketch a chromatogram of your expected result. Clearly label the axes in your chromatogram and also indicate which protein(s) are present in each peak. Pay careful attention to the intensities/areas of your peaks.Sketch an SDS-PAGE gel that includes the “precision plus protein” bio-rad ladder, a “pre-purification” sample, and samples that correspond to each of your resolved peaks in your chromatogram

Proteins are biomolecules, that is compose of amino acids linked by the peptide bonds. Each protein…

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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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You are given an equimolar (0.10 mM) mixture of Ubiquitin protein that is free/pure of any other macromolecules (e.g. nucleic acids) in a pH 7.0 phosphate buffer

How can you purify Ubiquitin using Ion-Exchange chromatography (IEC)? Propose a plan using steps to purify this protein.

Basic rules:

Affinity chromatography (e.g. His Tag purification) is not an allowed step because these proteins are in their native state (i.e. – do not have a polyhistidine tag).
Also, you are not allowed to rely solely on the color of certain proteins (e.g. cytochrome C and GFP) in your characterization/proposal.
You are allowed to buffer exchange (i.e. – switch buffers) but try to keep your pH’s in a reasonable range (5.5 – 8.5) or you risk denaturing your protein.
Assume proteins with the same charge (positive or negative) are not easily separated using IEC.
Assume all proteins can be resolved/visualized on an SDS-PAGE gel.

Ubiquitin Molecular weight (8663.02); pI (6.56); ε (M^-1 cm^-1) ignoring C’s (2980)

Describe your first proposed step clearly in words (1-2 sentences) including the buffer/pH used.
Sketch a chromatogram of your expected result. Clearly label the axes in your chromatogram and also indicate which protein(s) are present in each peak. Pay careful attention to the intensities/areas of your peaks.
Sketch an SDS-PAGE gel that includes the “precision plus protein” bio-rad ladder, a “pre-purification” sample, and samples that correspond to each of your resolved peaks in your chromatogram

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