Proteomics – 2D gels - Department of Chemistry and Biochemistry


20 Φεβ 2013 (πριν από 5 χρόνια και 5 μήνες)

173 εμφανίσεις


Its Function and Methods

Ryan Victor


What Is It?

What’s Being Studied?

How Is It Done?

Dimensional Electrophoresis

What is It?

Proteomics is the study of proteins that are
generated from the genetic code of an

While the genetic code for humans has
been completed through the human
genome project, proteomics will be a
much longer, more intensive and time
consuming study.

Proteomics differs from genomics in that the
chromosomes for a genome are consistent
throughout a multicellular organism, protein
output varies from cell to cell based on the
cells function.

What is It?

The study of proteomics is forced to focus on
very stringent conditions in a cell in order for
the results to be valid.

The cell must change its protein output
based on it’s level of stress and metabolic
needs. The results of a proteomics study
can really only be generalized to the
conditions that the cell faced during the

Initial attempts to study proteins used mRNA as
the determining factor for protein level output.
However, post translational modifications make
the method invalid.

What’s Being Studied

Phosphorylation: often is used to activate a
protein, usually on a serine or threonine residue.
Activation occurs as the phosphorylation is used
to signal other proteins and thus permit binding
between them.

Ubiquination: often is a signal for a protein to be degraded. Once a protein has
ubiquitin attached to it, it can be labeled for destruction by proteasome.

How to Study It

Antibody Method:

Antibodies are adding to the protein mixture

Antibodies bind to proteins that have modified

Proteins of interest can be separated based on the

2 Dimensional Protein Gels

What’s the Difference?

How’s It Done?

What Can We Do With It

Fluorescently labeled proteins in difference gel electrophoresis

The Difference

Conventional protein gels only run in one
dimension, as the name implies, 2D gels run in 2


running dimension allows separation
based on a 2


Common separation characteristics are
isoelectric point in the first dimension,
followed by mass/size separation in the


How It’s Done

We’ll look at isoelectric focusing as an example:

Proteins are fed into a gel medium that has a pH
gradient to it.

pH gradient is formed by adding polyampholytes to
the gel medium or using a gradient gel.

Polymapholytes are much like amino acids in that they
are zwitterionic.

How It’s Done

Proteins each have their own isoelectic
point that comes about as an average of
their amino acid isoelectic point.

Proteins are charged at different pHs.

When they reach the region of the gel that
matches their isoelectric point, they stop

Proteins can be removed at this point for
further analysis if desired.

Proteins can also be subjected to further
analysis within the gel.

How It’s Done

Now that the proteins have been separated by
isoelectic point, they can be analyzed based on
their mass.

Proteins are separated by mass using Sodium Dodecyl
Sulfate. SDS acts as a detergent to uncoil the protein
and give it a negative charge, since the proteins have
zero charge after the isoelectic focusing.

The proteins migrate by applying an
electric field 90 degrees from where
it was located for isoelectric focusing.

Proteins have now migrated in 2 different
dimensions from their starting point,
giving a “3D” gel.

How It’s Done

Now that the proteins have migrated to
their new positions based on isoelectric
point and mass, they can be analyzed.

Gel can be stained using coomassie or

Silver binds to the cysteine groups in
the protein, and leaves a dark stain on
the gel after development.

Coomassie binds to arginine, histidine,
and aromatic amino acids. It can also
be used to replace SDS to give the
negative charge to the protein.


Comparing the protein output between two different organism samples

Comparing output between cells within a specific organism

Determining whether an organism lacks a specific protein output

Associating protein output with a specific disease, and thus aiding in drug


Two dimensional gels must still be read, which has its own variety
of difficulties


Spots can overlap


Gel may not be reproducible


Spots may not properly visualize and be
too weak to see.

Scanners and computer programs are used to read the gels. They are only as
good as the scanners acuity and the programs ability to discern spots on the scan.

Duplicate gels overlaid upon each other in the
Delta 2D program

Difference Gel Electrophoresis

Fluorescent dye is added to the proteins,
then the gel is analyzed using a laser to
excite the fluorescent dyes.

Eliminates the difficulty of comparing
different gels.

The abundance of proteins can be
viewed from different samples to
give an idea of the difference
between them.


Proteomics allows researchers to study the actual output of
the cells rather than just the DNA blueprints.

Proteomics can be used to develop effective treatments for

Dimensional Electrophoresis is an effective method for
visualizing the results of a proteomics experiment.

2D gels are not without their difficulties.


Unlu, M., Morgan, M., Minden J. (1997). Difference Gel Electrophoresis.
11, 2071

King, M. (2009, November 2
). Protein Modifications. Retrieved March 5

2009 from

The Medical Biochemistry Page website,

Anderson, N. (2005). Proteome and Proteomics.
19, 1853

Maiman Institute for Proteome Research. Two
Dimensional Gel Electrophoresis. Retrieved

March 5

2009 from the Maimane Institute for Proteome Research at Tel Aviv website,

Berth, M. et al. (2007) The State of the Art in Analysis of Two
Dimensional Gel

Electrophoresis Images.
Applied Microbiology Biotechnology
76, 1223