Electrophoresis and Serological Testing
I.
INTRODUCTION
One of the most important facts you will learn in lecture is that blood serum or plasma contains
about 7 grams of protein per 100 ml (7 gm% or 7%). This protein will be referred to many times during
the course as a source of different physiologically important proteins and as the basis for the plasma
colloid osmotic pressure which will be discussed later in the course. The clinical laboratory is very
interested in being able to separate the serum protein into distinct observable bands that can be used to
diagnose various disease states.
In 1937, a Swedish biochemist, Tiselius, introduced the process of electrophoresis.
Electrophoresis is a fast, accurate way to separate these proteins. The basic principle of electrophoresis
is that serum protein molecules are charged with a negative charge and that these molecules will migrate
towards the positive pole (anode) in an electric field. Proteins are negatively charged because they
contain an abundance of amino acids with R groups containing carboxyl units (COOH, which are acidic
groups because they can give up their H+ ion). Amino acids such as aspartic and glutamic acids have
such 'R" groups. When these amino acids are in a basic pH medium, such as blood serum which has a
pH of 7.4 the "R" group dissociates the hydrogen ion (H+) and leaves the electron behind on the oxygen
of the 'R" group (COO-). If enough of the amino acids on a protein have this property, then the net
charge on the protein is negative. When electrophoresis is used for the separation of serum proteins, a
buffer of pH 8.8 is used to maintain the basic pH and therefore, the negative charge on the proteins.
II.OBJECTIVES
1. To become familiar with the fact that serum/plasma proteins are negatively charged.
2. To understand principles of electrophoresis as they pertain to the separation of
DNA, RNA, proteins such as serum/plasma proteins.
3. To understand the principles of enzyme-linked immunosorbant assays and their
advantages and limitations compared to western blotting.
III. CONCEPTS
DNA, RNA and proteins all have molecular groups that can cause the overall molecule to have a
net charge. For example, proteins have charges associated with the R groups of each amino acid that
make up the protein strand. Depending on the particular R group of each amino acid, it may carry a
negative charge, a positive charge or no charge at all. (Consult your textbook for the structure of the 20
amino acids, and determine which are acidic, basic or neutral amino acids.) The net charge on any protein
is the sum of all the charges displayed on that protein. Placing a protein in a buffer solution of a specific
pH may help to expose charges on R groups by dissociating or binding H+ ions. Similarly, placing DNA
or RNA into a basic buffer will cause the phosphate backbone to dissociate H+ ions and the DNA/RNA
molecule will have a net negative charge.
In electrophoresis, we make use of the net charge on DNA, RNA, or proteins to help us separate
or sort the DNA and RNA into various sized molecules or various proteins found in human serum. In the
case of serum protein electrophoresis (SPE), a small volume of a patient’s serum is placed on an
absorbent paper strip (nitrocellulose.) The strip is then placed in a chamber containing buffer of a
particular pH (8.8 in this case.) A voltage is applied across the chamber using a power supply hooked to
electrodes (wires) in the chamber. A voltage is applied, such that the anode (+ electrode) and the cathode
(- electrode) are at opposite ends of the absorbent paper strip. Negatively charged proteins move across
the strip toward the anode (by charge attraction.) Protein mobility is favored by the voltage applied to the
strip, but is hindered by the size of the protein (larger proteins move slower than smaller proteins of the
same charge.) Below are the major bands or peaks produced in serum protein electrophoresis of the
normal person.
The general principle that governs electrophoresis is given by the following formula:
Mobility of a molecule= applied voltage x net charge on the molecule
Friction of the molecule
The rate of movement of a molecule is increased by increasing the applied voltage or
increasing the net electrical charge of the molecule. The rate of movement decreases with
increasing friction caused by molecular size and shape. The net electrical charge of a molecule
also determines the direction it will migrate. A molecule with a net positive charge will migrate
toward the negative side of the chamber. The net negatively charged molecule will move
toward the positive side of the chamber.
DNA, RNA, and amino acids, have chemical characteristics that make them easy to
separate by electrophoresis. Their molecular weight is low and their physical size offers little to
no friction to decrease mobility. The amphoteric nature of amino acids may be exploited to great
advantage in electrophoresis. Amphoteric molecules may possess a net positive charge, a net
negative charge, or be electrically neutral depending on their environment.
The pH at which an amino acid exists in solution as a neutral molecule, a zwitterion, is called its
isoelectric point (pI). As zwitterions, the amino acid is electrically neutral and will not migrate
to either side of the electrophoresis chamber. As depicted below the pH at which an amino acid
exists determines its net electrical charge and the type of charge (+ or -).
If you increase the pH of a solution of an amino acid by adding
hydroxyl ions, the hydrogen ion is removed from the -NH3+
group. During electrophoresis, this amino acid would move
toward the anode (the positive electrode)
If you decrease the pH by adding an acid to a solution of an amino
acid, the -COO- part of the zwitterion picks up a hydrogen ion.
This time, during electrophoresis, the amino acid would move towards
the cathode (the negative electrode).
Each amino acid has its own isoelectric point so its net charge will vary with the pH of the
solution it is in. It should be noted that amino acids in their crystalline form are electrically
neutral (zwitterions).
INTRODUCTION: ELISA (Enzyme Linked Immunosorbant Assay)
The purpose of ELISAs is to detect specific protein antigens using antibodies. In laboratory
medicine, ELISAs are commonly used to screen serum samples from animals to detect
antibodies to a specific pathogen. In the research environment, ELISAs can be modified to
detect a protein of interest.
Method:
1. 96 well plates are coated with an antigen. Sites unoccupied by the antigen are blocked with a
blocking buffer to prevent non-specific binding of sample. Draw this below:
2. Serum samples are added and incubated to allow specific antibodies to bind to specific
antigen. Antibodies that do not recognize and bind specifically to the antigen remain
unbound. Draw this below:
Positive sample
Negative sample
3. Non-specific antibodies that do not bind to the antigen are washed away. Draw this below.
Positive sample
Negative sample
4. An enzyme labeled (e.g. alkaline phosphatase or horseradish peroxidase) secondary antibody
is incubated with the samples and binds tightly to sample antibody. Draw this below.
Positive sample
negative sample
5. Unbound labeled antibody is washed away and a colorimetric substrate is added. Draw this
below.
Positive sample
negative sample
6. The enzyme cleaves the substrate causing a color change of the substrate solution. The
intensity of the color is quantified using a spectrophotometer and is proportional to the
amount of antibody present in the sample. (Obviously, a standard curve would have to be
generated, could you think of how to do that?).
Positive sample
negative sample
Follow the instructions on the PhysioEx handout, provided on the next few pages.
The following link has a very nice animation of ELISA:
http://www.biology.arizona.edu/IMMUNOLOGY/activities/elisa/technique.html?
 Compare and contrast the ELISA and the Western Blot Technique.
 Which test do you think would be more specific, the indirect ELISA or the Western blot
technique? Why?
 Why are proteins generally considered to be negatively charged?
 What are “R” groups?
 How can proteins be manipulated so that the entire protein is as negatively charged as it
can be?
 Once a protein is negatively charged, how can a sample of proteins be separated
electrophoretically?
 What happens to the proteins as they “run” out on the gel?
 Did you know that pregnancy tests are a modified ELISA (capture ELISAs). Can you think of
how the antibodies are arranged to detect human chorionic gonadotropin, the “pregnancy
hormone?”
 If a woman used a standard pregnancy test, very early in her pregnancy, why could she get a
“false negative?”
 When might these tests be used for clinical purposes?