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.

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 2

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.

How gel electrophoresis works 3

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