Pharos university
Faculty of Allied Medical SCIENCE
Clinical Laboratory Instrumentation
(MELI-201)
Dr. Tarek El Sewedy
Lecture 4
Electrophoresis
And
Spectrophotometers
Intended Learning Outcomes
Students will learn :
1. Electrophoresis principles.
2. Electrophoresis techniques.
3. Spectrophotometry
Lecture content
Electrophoresis principles.
DNA Electrophoresis.
Protein Electrophoresis
Spectrophotometers
Electrophoresis
Electrophoresis
 A method of separating molecules from a mixture of similar molecules using electrical
current.
 The electric current is passed through a porous solid material called gel containing
the mixture, and each molecule travels through the gel at a different rate, depending
on its size.
 Gels are either Agarose (for DNA) or polyacrylamide gels (for Proteins) .
 Electrophoresis is most commonly used to identify DNA fragments and proteins
 It can be followed by a technique called blotting to specifically identify a particular
DNA segment (Southern blotting) or certain type of protein (Western blotting) or RNA
(Northern Blotting)
1. DNA electrophoresis
How gel DNA electrophoresis works 1
 First a gel is prepared, DNA Gels are made of Agarose, a seaweed extract
similar to gelatin. The finished gel has a consistency similar to very firm
jello. This consistency offers resistance to the pieces of DNA as they try to
move through the gel. The gel is prepared with wells at one end so that
DNA samples can be loaded into the gel.
How gel electrophoresis works 2
 Once the DNA samples are loaded onto the gel, an electric
current is applied to the gel. DNA is negatively charged due
to all the phosphate groups in the backbone of DNA. Thus,
DNA will move towards the positive electrode.
How gel electrophoresis works 3
 As the pieces of DNA move through the gel, they will meet with
resistance. Larger pieces of DNA will have more difficulty moving
through the gel than smaller fragments. Thus, larger fragments
will move slower than smaller fragments. This allows separation
of different DNA fragments depending on size difference.
Electrophoretic mobility of DNA
Summary of DNA electrophoresis
2. Protein Electrophoresis
 Movement of proteins in polyacrylamide gels is more complex.
Standard polyacrylamide gels (PAGE) separate the proteins down
the gel based on differences in the protein’s degree of electrical
charge, shape, and size.
 A type of treatment called denaturation is used in SDS
polyacrylamide gels (SDS-PAGE) . The proteins are heated in a
solution called sodium dodecyl sulfate or SDS. This causes the
proteins to have similar charges and shapes. Therefore, separation
is based on size. Again, smaller molecules move more quickly to the
Anode on the lower portion of the gel.
Protein Electrophoresis
Protein Electrophoresis Gels
Electrophoresis in clinical Labs
 The electrophoretic graph is the interpretation of protein
electrophoresis and includes a variety of proteins
Uses of DNA Gel Electrophoresis
 Gel electrophoresis is used to provide genetic information in a wide range
of data fields. Human DNA can be analyzed to provide evidence in
criminal cases, to diagnose genetic diseases, and to solve paternity
cases. Samples can be obtained from any DNA-containing tissue or body
fluid, including tissue cells, blood, skin, hair, and semen. In many
analyses, polymerase chain reaction (PCR) is used to amplify specific
regions of DNA that are known to vary among individuals. A person’s “DNA
fingerprint” or “DNA profile” is constructed by using gel electrophoresis to
separate the DNA fragments from several of these highly variable regions.
Spectrophotometers
 Spectroscopy uses light to identify and determine the concentration of a
particular chemical in a solution.
 The term spectroscopy refers to the observation (scopy) of various wavelengths
of light (spectro).
 It makes use of the ultraviolet, visible, and infrared regions of light spectrum.
 Humans perceive different types of light as color. However, scientists measure
light as wavelength.
 Spectrophotometers are found in every clinical laboratory. They are
important for identifying biological molecules. These instruments are also
useful for determining the concentration and purity of almost all biological
molecules.
 The basic spectrophotometer is composed of a light source,
monochromator, sample holder, photodetector, and readout.
 Most spectroscopy uses a light source made up of a lamp that produces
visible light very much like a household light bulb. These lamps provide white
light which is made up of the whole visible light spectrum.
 Infrared or ultraviolet light lamps can also be used as a light source. The type
of light used depends on the nature of the chemical testing being performed.
 Lamps usually have to be warmed up and maintained at a certain
temperature to ensure consistent lighting during each use.
 An improperly operating lamp can give the wrong information without the
operator being aware of any errors
 spectrophotometers provide a pure beam of light consisting of a few
wavelengths that is then transmitted to the chemical sample.
 This pure beam of light is produced by using a device called a
monochromator.
 A monochromator is an adjustable crystal, filter, or mirror that isolates
portions of the light spectrum by separating the light into its component
wavelengths.
 The range of wavelengths provided by a monochromator is called the
bandwidth.
 The sample holder is an opening in the spectrophotometer
that places the sample between the beam path and the
photodetector.
 The sample holder grips a special container called a
cuvette. Cuvettes are made of glass, plastic, quartz. The
type of cuvette being used is determined by the
wavelength of the beam. Ultraviolet light spectroscopy
requires quartz cuvettes because glass and plastic
interfere with the passage of ultraviolet light to the sample.
 The photodetector, measures light:
1. Absorbance: amount of light absorbed by the sample.
2. Transmittance: amount the light passed through the sample.
3. Fluorescence: amount of glow given off by a sample that is
exposed to a particular wavelength of light.
 The photodetector converts the light into an electrical signal
that is displayed as a measurement on the readout.
 The readout usually displays the measurement as a number.
Assignment
 Abdulrahman Samir: is selected to make the assignment Different
applications of Electrophoresis
 The Assignment should be delivered before next lecture
Study questions
 Mentions 3 different applications of Electrophoresis
 Mention the main difference between DNA and Protein electrophoresis
Suggesting reading
 Encyclopedia of Medical Devices and Instrumentation, 2nd ed. New York:
Wiley, 2006