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

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
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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)

 

  1. Describe your first proposed step clearly in words (1-2 sentences) including the buffer/pH used.
  2. 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.
  3. 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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