Electrophoresis & Spectrophotometry

Guided By: Dr. Vishwa Prakash Shetty Dr. Aparna Dave. Dr. Manpreet Arora Dr. Radhika Rai Dr. Pulin Saluja

Presented by : Dr. Anshum Dutta M.D.S. 2nd year

Contents

Introduction to electrophoresis History Principle of electrophoresis Methodology Blotting Spectrophotometer Components History Principle of spectrophotometry. Procedure . Bibliography.

Introduction

In 1990 Human Genome Project, was formally begun under the sponsorship of the U.S. Department of Energy and the National Institutes of Health. The aim of this project was to extract the sequence of DNA . For this Sanger method was used.

Sanger method
The section of DNA to be examined known as the template is mixed with a segment of DNA that binds to part of this sequence.

This mixture is placed into four containers that have the nucleotides and enzyme needed to build on the template.

The DNA strands formed in each container are later separated according to their size. By comparing the length of these strands and by knowing which labelled nucleotides are at the end of each strand, the sequence of the DNA can be determined.

The Sanger method was originally developed as a manual technique but took long periods of time to perform.

Both traditional and newer systems for accomplishing this sequencing utilize a separation method and later is known as

ELECTROPHORESIS.

Electrophoresis

Electro = flow of electricity, & Phoresis, from the Greek = to carry across

Electrophoresis can be described as migration of particles under the influence of charged particles.

History

Separation of serum proteins by electrophoresis was first attempted by Tiselius in 1937.

In his experiment the proteins moved to the oppositely charged electrode in free solution. Such free electrophoresis, however, suffers from one important disadvantage:
As passage of electric current produces heat that brings about convection flow of the liquid medium, which, in turn, disturbs the zones of separated proteins.

Therefore, nowadays electrophoresis is usually performed in some support, which can be paper, cellulose acetate, or gel made of starch, agarose or polyacrylamide.

Principle

At any given pH either in solution as electrically charged species. either as anions (-) or cations (+) Under the influence of an electrical field these particles migrate to the cathode (negative electrode) or anode (positive electrode), depending the nature on of their net charge. Many important biological molecules such as amino acids. peptides, proteins, nucleotides and nucleic acid, possess ionizable groups. So, electrophoresis is frequently utilized in biochemical and medical research.

Factors affecting migration of charged particles.

1. 2.

Charge : Greater the charge on a particle, greater the

distance migrated by charged particles.

Time : increase in time of electrophoresis, the distance

migrated by charge particle can be increased. 3. Voltage : By increasing the applied voltage the migration distance of the charged particles can be increased.
4.

Distance between the electrodes: When

distance between the electrodes is increased, distance migrated by the charged particle decreases due to reduction in effective voltage.

5.

Ionic strength of the buffer : When the ionic

strength of a buffer increased , decrease in the is migration of charged particles takes place.

6.

pH of buffer : the direction of movement of charged

particles depends upon the pH of the buffer used for electrophoresis. Eg: at pH 8.6 all serum proteins bear a net negative change & move towards the anode. If the pH of the buffer is adjusted below 4.8, then all the proteins will move towards the cathode, since they carry positive charge.

7.

Size of the molecule : Speed of the movement of

charged particles is dependent upon the size of the charged particles. When applied net charge is equal, small particles carry more charge & move at faster rate & large particles carry less charge & move at slower rate.

How Is Electrophoresis Performed?

Electrophoresis can be performed in a variety of formats

By applying small amount of a sample to a support (usually a gel) and allow the this sample to travel in a running buffer through the support when an electric field is applied. This approach is known as gel electrophoresis. . By separating the components of a sample by using a narrow capillary that is filled with a running buffer and placed into an electric field. This second format is called capillary electrophoresis.

Gel electrophoresis

In this type of system, several samples are usually applied to the gel and allowed to migrate along the length of the support in the presence of an applied electric field.

The farther this distance is from the point of sample application, the higher the migration velocity the larger its electrophoretic mobility. In the case of gel electrophoresis, the separation is stopped before analytes have travelled off the support.

This migration distance will, in turn, be related to the size and charge and can be used in identifying such a substance.

The result is a series of bands where the migration distance (dm) characterizes the extent to which each analyte has interacted with the electric field.

Components

Power supplier. Buffer tank fitted with electrodes with a support to position the support medium & insulating transparent cover.

 Buffer ( Tris/acetate/EDTA) (Tris/borate/EDTA) Staining solution (Bromophenol blue) Densitometer

Densitometer

A densitometer is a device that measures the degree of darkness (the optical density) of a photographic or semitransparent material or of a reflecting surface. It determines the density of a sample placed between the light source and the photoelectric cell from differences in the readings.

How does it work?

DNA is negatively charged.

When placed in an electrical field, DNA will migrate toward the positive pole (anode).

An agarose gel is used to slow the movement of DNA and separate by size.

Support media
Paper. Agar and Agarose. Cellulose acetate. Polyacrylamide.

Paper Electrophoresis
Filter paper ( 1mm or 3 mm) is used. It was earliest supporting media but now been replaced by other supporting media. Separation of components takes long time ( 14 to 18 hrs.)

Agar & Agarose

Both used as supporting media. Agar is made of two constituents : agaropectin & agarose. Agaropectin has galactosesulfate & carboxylic group, which are responsible for significant electroendosmosis associated with agar gels. Agarose is agar without agaropectin molecule.

Agarose has large pore size & used as an ideal supporting medium for electrophoretic separation of serum protein, lipoproteins, haemoglobins & isoenzymes. Electrophoresis time is relatively rapid , require 30 to 120 minutes. Being transparent, agarose gel can be readily scanned with densitometer.

Cellulose acetate

This supporting medium is delicate compared to paper & requires careful handling . The various advantages of this medium are :
It

shows minimum adsorption & clear separation of a mixture into discrete zones.
separation of specimen components can be completed in 30 to 60 minutes.

The

Polyacrylamide gel electrophoresis

(PAGE)

It is a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, and can be prepared with a wide range of average pore sizes. Separation of various components in a mixture or sample can be performed both on the basis of molecular charge & size. SDS-PAGE is one of most commonly used technique in research labs.

Sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein sample to linearize proteins and to impart a negative charge to them, so that they may not have secondary or tertiary structure.

Blotting

Another possible approach for detection in gel electrophoresis is to transfer a portion of the analyte bands to a second support (such as nitrocelluose), where they are reacted with a labelled agent. This approach is known as blotting.

A Southern blot is used to detect specific sequences of DNA by having these sequences bind to an added, known sequence of DNA that is labelled with a radioactive tag or with a label that can undergo chemiluminescence. A Northern blot is similar, but is instead used to detect specific sequences of RNA by using a labelled DNA probe.

Another type of blotting method is a Western blot. A Western blot is used to detect specific proteins on an electrophoresis support. In this technique, proteins are first separated on a support by electrophoresis and then blotted onto a second support like nitrocellulose or nylon. The second support is then treated with labelled antibodies that can specifically bind the proteins of interest.

After the antibodies and proteins have been allowed to form complexes, any extra antibodies are washed away.
Remaining bound antibodies are detected through their labels.

This method is used to screen blood for the HIV virus by looking for the presence of proteins from this virus in samples.

Capillary Electrophoresis
It separates analytes by electrophoresis and that is carried out in a capillary. This method was first reported in the late 1970s and early 1980s and is sometimes known as capillary zone electrophoresis.

The use of these narrow-bore tubes provides efficient removal of Joule heating by allowing this heat to be quickly dissipated to the surrounding environment. This removal of heat helps to decrease bandbroadening and provides much more efficient and faster separations than gel electrophoresis. The capillary in a CE system is typically made of fused silica.

Application of electrophoresis:

Sequencing of DNA. Purification of proteins, peptides, and other bio molecules. For examining the patterns of amino acids, serum proteins, enzymes, and lipoproteins in the body. Used in the analysis of organic and inorganic ions in foods, commercial products, and environmental samples.

For the characterization of proteins in normal and diseased cells and for looking for new substances.
EPD is used in industry for the coating of primers on car bodies.

Electrophoretic deposition (EPD) was used as shaping technique for ceramic dental crowns and dental three-unit and four-unit bridges in the high loaded molar region.

For EPD plaster stumps which had been prepared for the dental crowns and bridges by the dental technician were coated with silver and were directly used as deposition electrodes.

T. Moritz, W. Eiselt, K. Moritz . Electrophoretic deposition applied to ceramic dental crowns and bridges. Journal of Materials Science .December 2006 :41 :24 , 8123-8129

The single cell gel electrophoresis (SCGE) assay, also known as the comet assay, is a cytogenetic technique for measuring and analyzing DNA single stranded breaks (SSB) and/or alkali labile sites within individual cells.
Peripheral blood leukocytes of 22 oral squamous cell carcinoma (OSCC) patients were subjected to SCGE and the DNA damage levels (SSB) were quantified with respect to clinical staging and histopathological grading.

G. Venkateswara Rao. G. S. Kumar Y. R. Ahuja. Single cell gel electrophoresis on peripheral blood leukocytes of patients with oral squamous cell carcinoma 1997 .26 :8 : 377380,

Highly statistically significant differences in DNA damage levels were found between normal subjects and patients with OSCC of the same age group. DNA damage levels were altered in all clinical stages and histopathological grades of oral squamous cell carcinoma. The results support the concept of a systemic host response in malignancy.

G. Venkateswara Rao. G. S. Kumar Y. R. Ahuja Single cell gel electrophoresis on peripheral blood leukocytes of patients with oral squamous cell carcinoma 1997 .26 :8 : 377380,

Procedure
1.

2.

Gel is poured in plastic casting tray & after the gel solidifies comb is removed. Size of gel prepared is 17cm x 43cm x 0.4cm. Fill the electrophoresis tank with 1X TBE buffer & then mount the gel in it.

Apparatus is Pre- runed at 60W for 30-45 min in order to warm the gel up to a plate temperature of 45C 50C. PCR amplified sample is loaded on the gel & separated for 60-90 min. Sample is prepared by mixing 2l of the amplified sample with 3l of bromophenol blue & glycerol.

Gel is then removed & stained with ethidium bromide & observed under UV illumination.

Hazards of Electrophoresis

Toxicity : DNA visualization uses dyes like Ethidium Bromide which chelate DNA and is a known carcinogen. Although some safer alternatives are becoming available (SYBR Safe). Polyacrylamide, used as a matrix in protein gels is also a known neurotoxin.
Accuracy : Unless densitometry is used to measure the density of each protein or DNA band, Gels can't really give an accurate idea of the concentration or abundancy of protein or DNA.

Spectrophotometry

Spectro' means band of colours, 'photo' means light and 'meter' is a device for measuring something.
Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.

Spectrophotometer functions similar to photo colorimeter. The selected light rays by prism is made to pass through a silt thus making a particular wavelength to strike the sample cell & then passes to photo detector.

History

The spectrophotometer was invented in 1940, by Arnold J. Beckman and his colleagues at National Technologies Laboratories,
Before 1940, the chemical analysis process was a long venture taking weeks to complete with only 25 percent accuracy.

In 1981 microprocessor controlled spectrophotometer were introduced. This automated the device and improved the speed.
With the 1990s came the addition of external software that provided PC control and onscreen displays of the spectra.

Principles

The working of spectrophotometers is based on Beer & Lamberts law.

Beers

Law : It states that the optical density of a solution is directly proportional to the concentration of the solution. Lamberts law : It states that the optical density of a coloured solution is directly proportional to the path of light.

So according to beer & Lamberts law the absorbance of a solution containing light absorbing material, depends on the following factors.
The

nature of substance. The wavelength of the light. Path of light The amount of coloured material in the light path.

In other words, as the colour of the solution deepens, as its concentration also increases & which is measured by spectrophotometer. This is an underlying principle of spectrophotometry.

Components

Light Source: Spectrophotometer has two light

source. Deuterium light source is used for UV region and tungsten lamp used for visible light region.

Monochromator: Monochromator divides the incident

light into their component wavelength. This can be done by using prism (or) differing grate Monochromatic light can still be purified by passing through a sit lenses, filter and mirrors.

Photodetector: Photodetector converts light energy

into electrical signals.

Cuvette

A cuvette is a small tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz (for UV light) and designed to hold samples for spectroscopic experiments.

Light Source

Tungsten Lamp: Tungsten Halogen Lamp, it is the

most common light source used in spectrophotometer.

This lamp consists of a tungsten filament enclosed in a glass envelope, with a wavelength range of about 330 to 900 nm, are used for the visible region.

They are generally useful for measuring moderately dilute solutions in which the change in colour intensity varies significantly with changes in concentration .

Hydrogen / Deuterium Lamps: For the ultraviolet region, hydrogen or deuterium lamps are frequently used Their range is approximately 200 to 450 nm. Deuterium lamps are generally more stable and has long life about 500h.This lamp generates continuous or discontinuous spectra.

Xenon flash lamps : Xenon flash lamps have several advantages as the following : 1)Their range between ( 190nm - 1000 nm) 2) Emit both UV and visible wavelengths 3) Long life 4) Do not heat up the instrument 5) Reduce warm up time.

Procedure

Place sample into cuvette using a pipette. Use new pipette tip for every sample. Wipe the cuvette with very fine paper to remove any water droplets or dust. Avoid scratching the cuvette. The cuvette should never be overfilled 2/3 full is sufficient. Place cuvette into spectrophotometer.

Take a reading. Remove cuvette and discard liquid into beaker with waste water. Rinse the cuvette with deionized water 3 times before placing another sample. Repeat the procedure.

There are two classes of spectrophotometers: 1)Single beam:

All the light passes through the sample. In this case, to measure the intensity of the incident light, the sample must be removed so all the light can pass through. This type is cheaper because there are less parts and the system is less complicated.

2)Double beam :
The double beam instrument design aims to eliminate errors by measuring blank and sample virtually simultaneously. To measure a spectrum with a double beam instrument the two cuvettes, both containing solvent are place in the sample and reference positions

Advantage High stability because reference and sample are measured virtually at the same moment in time. Disadvantage Higher cost, lower sensitivity because throughput of light is poorer because of the more complex optics and lower reliability because of the greater complexity.

Split Beam:
The split beam spectrophotometer is similar to the double beam spectrophotometer but it uses a beam splitter instead of a rotating disc to send light along the blank and sample paths simultaneously to two separate but identical detectors.

Blank and sample measurements can be made at the same moment in time.
Spectra are measured in the same way as with a double beam spectrophotometer.

Beam splitter

A beam splitter is an optical device that splits a beam of light in two. In its most common form, a cube, it is made from two triangular glass prisms which are glued together at their base using polyester, epoxy, or urethane-based adhesives.

Advantage

Good stability, though not as good as a double beam instrument because two detectors can drift independently.

Application of spectrophotometery

In Forensics for determining facts and evidences. Textile industries, manufacturers of printers, and ink manufacturers.

Determining the concentration of DNA and RNA.

For estimation of drug content in pharmaceutical products.

Hazards associated with the use of UV/ VISIBLE spectrophotometers.

Potentially dangerous ultra violet radiation is emitted from the ultra violet lamp. Do not open the lamp housing while the UV lamp is in use.

Bibliography

Medical Laboratory technology. Volume 1, Ramnik sood. Medical Laboratory technology. M. Muthuprasanna. Textbook of biochemistry. David E. Metzler. Textbook of biochemistry. U. Satyanarayana. Forensic DNA Typing. John M. Butler. 2nd edition. T. Moritz, W. Eiselt, K. Moritz . Electrophoretic deposition applied to ceramic dental crowns and bridges. Journal of Materials Science .December 2006 :41 :24 , 8123-8129 G. Venkateswara Rao. G. S. Kumar Y. R. Ahuja Single cell gel electrophoresis on peripheral blood leukocytes of patients with oral squamous cell carcinoma 1997 .26 :8 : 377380

Shapiro AL, Viuela E, Maizel JV Jr. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 28 (5): 815820 L. Stappers , L. Zhang, O. Van der Biest, J. Fransaer. The effect of electrolyte conductivity on electrophoretic deposition. Journal of Colloid and Interface Science. 328 (2008) 436446.