ACKNOWLEDGEMENT

Before getting into the details of the project, i would like to express my wholehearted gratitude to one and all , who have helped me in
doing this project successfully.

First of all, i thank my biology teacher Mrs.bindhu and biology lab assistant Mrs. Prema for their support , guidance and their valuable
advices.

I express my sincere thanks to my group members, Vijay P and Azhar UP, for their valuable contributions for this project.

Iam indebted to my parents and sister for thier constant encouragement and support

Finally, i would like to thank CBSE for giving me this opportunity to undertake this project.

Above all, i thank Almighty for making this venture a success.

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Contents

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Introduction

Electrophoresis is a simple, rapid and highly sensitive tool to analyze proteins. The separation of proteins by electrophoresis is based on
the fact that charged molecules will migrate through a gel matrix upon application of an electric field

. Ions run more or less quickly along the substrate according their charge, size, shape, etc. Electrophoresis is
widely used to separate substances such as amino acids, proteins, strands of DNA, etc. As in the case of
chromatography, people use different supports and solvents according to the substances to be separated and
the techniques used

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Paper electrophoresis is useful for the separation of small charged molecules such as amino acids and small proteins. A strip of
filter paper is moistened with buffer and the ends of the strip are immersed into buffer reservoirs containing the electrodes.
The samples are spotted in the center of the paper, high voltage is applied, and the spots migrate according to their charges.
After electrophoresis, the separated components can be detected by a variety of staining techniques, depending upon their
chemical identity.

Electrophoretic techniques have also been adapted to other applications such as the determination of protein isoelectric points.
Affinity gels with biospecific properties are used to study binding sites and surface features of proteins. Continuous flow
electrophoresis is applied to separations in free solution and has found very useful application in blood cell separation.
Recently, High Performance Capillary Electrophoresis (HPCE) has been developed for the separation of many classes of
biological molecules

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Paper electrophoresis attained widespread use in the 1950s. It is a scientific method of separating a substance to determine its
composition. Wide-ranging ramifications of this method and resulting newer electrophoresis technologies have served scientific
and other industries as well..

Scientific Applications

The main use of paper electrophoresis is that of analysis. Scientists have used this technique in many scientific
experiments as a way to prove or disprove hypotheses or to determine the efficacy of drug treatments. ,
scientists use a specific paper electrophoresis test, called the Western blot, to detect the presence of human
immunodeficiency virus (HIV) in blood samples. In addition, other blot tests help detect the type and amount of
DNA and RNA in sampled materials, which has major implications for forensic investigations. Furthermore,
paper electrophoresis can be used for testing suitability of municipal water supplies, toxicity of waste water and
other environmental components.

Drug Testing
 The drug-testing industry has used paper electrophoresis to determine the presence of illegal or recreational
drugs in job applicants and crime suspects. Furthermore, sports authorities such as the International Olympic
Committee have used the technology to screen athletes suspected of ingesting illegal performance-enhancing
supplements.

Ink Analysis

Inks such as those used to print currency and checks consist of diverse chemical makeups. Investigators and
forensic scientists have used paper electrophoresis since the 1950s to analyze inks. Results of these tests help
determine the authenticity of suspected counterfeits, forgeries and other fraudulent documentation.

E-Paper

E Ink used electrophoretic technology in pioneering electronic paper display (EPD) technology. The company
invented an electronic ink that consists of millions of minute microcapsules, about 100,000 per square inch of
paper, containing a clear fluid in which positively charged particles of one color and negatively charged particles
of a contrasting color reside. When an electric field is applied to a paper or film printed with the ink, all the
particles of one charge rise to the surface, depending on the polarity of the electricity

This technology is being used for watches, mobile phones and other handheld electronics, as well as for signage. The low
voltage required makes these items tremendously inexpensive to operate.

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Aim

To obtain the blood components , in the anode of electrophoresis apparatus, by the technique of paper
electrophoresis and also to scrap out the blood smear in the anode to observe under the high power of a
microscope.

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Apparatus required

two small basins

a support (i.e.: filter paper, cellulose acetate strips, polyacrylamidegel, or a capillary tube)

, an electrical DC power supply

two electrodes

table salt

baking soda

saffranin

600ml water

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procedure
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initial procedure

1. place two little basins a few cm (one inch) apart.

2. Pour in the basins an electrolyte made of a teaspoon of table salt and another of baking soda in 300
ml(1 1/2 cups) of tap water
3. . Place a glass plate on the two basins and on it place a strip of filter paper soaked with the
electrolyte. This strip has to be immersed with each end in the electrolyte in the basin for to complete
the electrical circuit
4. . With a pencil, draw a line across the filter paper and place a little drop of blood on it
5. . Cover the paper with a second glass plate
6. . Put an electrode into the electrolyte of each basin and apply 45V in direct current . You can obtain
this from five, 9volt batteries connected in series.
7. Observe the system after two hours

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Observations

With the passing of time,it is observed that, different little white coloured spots are moved towards the
negative electrode.

Explanation

At pH 7 basic amino acids will be positively charged and so they will behave as cations. Acidic amino acids will be
negatively charged and so they will behave as anions. Neutral amino acids will be both positively and negatively
charged (zwitterions) and so they will stay in the centre. These amino acids are said to be at their isoelectric point.
Each type of amino acid has its isoelectric point at a particular pH, it depends upon the number of ionisable groups in
the molecule. At any pH above the isoelectric point the molecule will have a net negative charge and move towards the
anode. Similarly at pHs below the isoelectric point the molecule will have a net positive charge and move towards the
cathode.

The relative charge on each species of amino acid will determine the speed and direction of travel. Thus they can be
separated.

As proteins are made of amino acids and amino acids migrate in an electric current, then proteins too will migrate
(slowly). The speed and direction of migration will depend upon the numbers of the characteristic amino acids in the
protein's structure. A protein made up of a lot of basic amino acids will not behave in the same way as a protein made
up of acidic ones, they will have different isoelectric points

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Final procedure

1. The white spots accumulated at the anode are scrapped with a blade to a slide
 2. Add a small drope of saffranin and keep a coverslip over it
3. Observe it, first under low power and then under rhe high power of microscope

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Observations

Under the high ower of microscope, the blood components are observed as greenish blue patches of proteins
spread over the red background of RBCs

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Result

The greenish blue patches observed are made of different protein components of the plasma (serum
proteins): globulins (alpha, beta, gamma), albumin and fibrinogen. These are aggregated together into small
patches under the action of electrolytic movement through the strip of filter paper.

Blood is a highly specialized tissue composed of many different kinds of components.  Four of the most important ones are red
cells, white cells, platelets, and plasma.  All humans produce these blood components--there are no populational or regional
differences.

The main protein components of the plasma are:

1 Serum albumin, often referred to simply as albumin is the most abundant plasma protein in mammals. Albumin is essential for
maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular compartments and body
tissues
2 Globulin is one of the three types of serum proteins. Some globulins are produced in the liver, while others are made by the
immune system.they are of 4 types-

*Alpha 1 globulins -

*Alpha 2 globulins

*Beta globulins

*Gamma globulins

3 Fibrinogen (factor I) is a a soluble plasma glycoprotein, synthesised by the liver, that is converted by thrombin into fibrin during
blood coagulation

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Precautions

1. Do not use high voltages for this experiment.2.

2. Never touch the electrodes, the electrolytes, or the paper strip.
3. Take care never to short out the two electrodes.
4. insert a fuse on one of the two cables. The fuse will break the circuit when there is too high a current.

. At the end of the migration of the spots, remove the electrodes from the basins and take off the cables from
the batteries.

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KEY TERMS

1. Biological activity
—The specific biochemical function of a biomolecule such as enzyme affinity for a substrate.
2. Biomolecule
—An organic compound present in living organisms.
3. Electrophoresis gel
—A natural or synthetic polymer matrix used as a support for sample separation in electrophoresis.
 4. Isoelectric point
—The pH at which a molecule has no electric charge and does not move in an electric field.
5. Molecular sieving
—The restriction of molecules migrating through a gel due to their size

Bibliography

 Icbse.com
 Wikipedia
 Science.jrank.org
 Ehow.com