BIO 301L. 1st semester 2025-2026. ATGeron
Laboratory Activity No. 9
Protein Separation by Polyacrylamide Gel Electrophoresis (PAGE)
Electrophoresis is a principal biochemical tool used to characterize, separate, and purify
charged macromolecules such as proteins.
Proteins differ in their molecular weights and net charges based on the number and type of
amino acids present in their polypeptide chains. However, the net charge of a protein is dependent on
its environmental pH, such that there exists a certain pH at which a given protein exhibits no charge.
This is called its isolectric point. Therefore, by placing different proteins in a solution with a pH other
than their isoelectric points, each protein can be separated by applying an electric field. Depending on
their net charges and molecular weight, these proteins will differentially migrate to the electrode they
are attracted to. The greater the net charge of the protein, the faster is its rate of migration towards one
of the poles and vice versa. The larger the protein the more difficult it is for it to pass through a
supporting medium and vice versa.
Electrophoresis uses various supporting media where macromolecules migrate. These may be
cellulose, cellulose acetate, alumina, and gels of agarose, polyacrylamide, silica, and starch. These
materials form spaces (or pores) in between their molecules that serve as molecular sieves to the
proteins that would be passing through them. In the case of gels, pore sizes can be easily changed by
altering its concentration, such that increasing the concentration of the gel results in a decrease in its
pore size. These gels may be run either horizontally or vertically.
Of the different electrophoretic media available, polyacrylamide is one of the more popular in
use. This is because polyacrylamide gel has a number of advantages over other gel types, namely
(a) it is chemically inert, (b) it is stable over a wide range of pH, temperature, and ionic strength,
(c) it is transparent, and (d) its pore size can vary over a wide range.
A polyacrylamide gel is the result of the polymerization of acrylamide monomers into long
chains, and the crosslinking of individual chains with bifunctional compounds, such as N,N’methylene
bisacrylamide. Figures 1 and 2 illustrate the structure of acrylamide and busacrylamide monomers and
the polyacrylamide gel. The polymerization process is initiated by the addition of ammonium
persulfate (APS) or riboflavin. Aside from this, N,N,N’N’- tetramethylenediamine (TEMED), when
added, acts as an accelerator of polymerization. However, oxygen inhibits polymerization; thus, the
gel mixtures are usually degassed before the addition of APS and TEMED.
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BIO 301L. 1st semester 2025-2026. ATGeron
(a)
(b)
Figure 1. The chemical structures of (a) acrylamide and (b) N,N’-methylene bisacrylamide.
Figure 2. Crosslinking of acrylamide and bisacrylamide.
INTENDED LEARNING OUTCOMES
1. Discuss the principle of polyacrylamide gel electrophoresis
2. Familiarize with the method of separating proteins using PAGE
3. Explain the rationale behind the steps in PAGE.
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BIO 301L. 1st semester 2025-2026. ATGeron
MATERIALS
PAGE videos and protocols (Part II: Methods)
METHODS
1. Read and understand the module and protocol (Part II: Methods).
2. Read
the
protocol:
https://www.med.unc.edu/pharm/sondeklab/wp-
content/uploads/sites/868/2018/10/Native-gel-analysis.pdf
3. Watch the videos assigned
SDS-PAGE Animation: https://www.youtube.com/watch?v=i_6y6Z5UvwE
SDS-PAGE: https://www.youtube.com/watch?v=bdBXwuuwSBo
SDS-PAGE playlist: https://www.youtube.com/playlist?list=PL3264CF81D53FD4B8
Western blotting: https://www.youtube.com/watch?v=VgAuZ6dBOfs
Answer the questions in the worksheet
Reference:
Lodish B, A Berk, CA Kaiser, M Krieger, A Bretscher, H Ploegh, A Amon, and KC Martin. 2016.
Molecular Cell Biology. 8th ed. New York: W.H. Freeman.
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