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? Propose a 2-step minimum plan to purify their 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 you can only separate proteins under 50.0 kDa with a 5.0 kDa difference using SEC. Otherwise, assume proteins greater than 50.0 kDa are unable to be separated. 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…

Answered: Describe your first proposed step…

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? Propose a 2-step minimum plan to purify their 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 you can only separate proteins under 50.0 kDa with a 5.0 kDa difference using SEC. Otherwise, assume proteins greater than 50.0 kDa are unable to be separated.
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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