# Proteomics/Protein Separations - Chromatography

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Chapter written by: Laura Grell and Alexander Butarbutar\
Contact llg3875@rit.edu or nbb3924@rit.edu for contributions\
Chapter modified by Kai Burnett and Dalia Ghoneim\
Contact kab9783@rit.edu or dxg6098@rit.edu \_\_FORCETOC\_\_

## Introduction

!HPLC{width="350"}

(Res1)
To obtain a pure protein sample, a protein must be isolated from all
other proteins and cellular components. This can prove to be a difficult
task as a single protein often makes up only 1% of the total protein
concentration of a cell. Therefore 99% of the protein components of a
sample must be removed before it can be classified as pure. A task that
is equally challenging is keeping the protein in its active form. When
we purify proteins we remove them from their natural environments. As a
result, it is necessary to simulate the pH, salt concentration and
reducing conditions in which they are normally found. In the process of
obtaining an active and pure sample we want to minimize the number of
steps taken in order to maximize the yield at the end of the separation.
Finally, since proteins are made with the intention of only functioning
for a short period of time, it is also critical to obtain our sample as
quickly as possible. All these components of protein separations can be
successfully achieved by a group of separation methods collectively
known as
chromatography.

There are several properties of proteins that can be taken advantage of
to separate proteins. Different types of chromatography take advantage
of different properties. Proteins can be separated by:

-   size
-   shape
-   hydrophobicity
-   affinity to molecules
-   charge

!A Typical Column{width="350"}    In
this chapter several different chromatographic methods will be
introduced and described. While the methods outlined below all use
different characteristics of proteins to separate proteins from one
another, they all utilize an insoluble stationary phase and a mobile
phase that passes over it. The mobile phase is commonly a liquid
solution. It contains the protein we want to isolate. The stationary
phase on the other hand is made up of a grouping of beads, usually based
on a carbohydrate or acrylamide derivative, that are bound to ionically
charged species, hydrophobic characters, or affinity ligands. Much of
the success of chromatography is associated with the selection of an
appropriate stationary phase.

   In column
chromatography,
when a protein sample is applied to the column, it equilibrates between
the stationary phase and the mobile phase. Depending on the type of
chromatography, proteins with certain characteristics will bind to the
stationary phase while those lacking the sought characteristics will
remain in the mobile phase and pass through the column. For example in
ion exchange chromatography, a positively charged protein would bind to
a negatively charged stationary phase, while the negatively charge
protein will be eluted from the column with the mobile phase. The final
step involves displacing the protein from the stationary phase, also
known as elution, by introducing a particle which will compete with the
protein binding site on the stationary phase. Today various commercial
column are readily available, specifically
Bio-Rad,
Sigma-Aldrich,GE
Healthcare
and Omnifit offers a
wide variety of chromatography column.

!Common Chromatogram RIT Ion Exchange
Site


The image above is a chromatogram that shows the results of a separation
based on signals interpreted by a detector.

t~m~ - the time required for the mobile phase to travel the entire
length of the column

t~r~ - the time required for a specific protein to elute from the column

## Resources

1.  Craig, P. Designing a
    Separation
2.  Florida State University, \"Chromatography\" Michael Blaber\'s
    Biochemistry
    Lab
3.  GE Healthcare - 1 -
    \[<http://www.gelifesciences.com/protein-purification>?\]
4.  BioPharm International Guide Basics of
    Chromatography
    (2003).
5.  Bio-Rad Chromatography Protein
    Purification
    \|\| Green Fluorescent Protein Chromatography
    Kit
6.  BioForum Topics In
    Chromatography
7.  Journal of Chromatographic Science
8.  M.Isabel Pedraza Mayer Chromatography
    Database

## References

1.  Harris, D.C. \"Quantitative Chemical Analysis; 6th Edition\", W.H.
    Freeman and Company: New York..
2.  Patrick McKay An Introduction to
    Chromatography
    Senior Research Associate, Department of Recovery Sciences,
    Genentech, Inc \*.

.\* Denotes Free Article

