1. Gel Electrophoresis
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Name : Abhishek Chauhan
Class : 1st SEM M.Pharm
Department : Pharmaceutics
Submitted to –
Dr. Pooja Chawla
(HOD of Chemistry and Analysis
Department)
2. ELECTROPHORESIS
• Electrophoresis is the separation of charged molecules in an applied electric
field.
• The relative mobility of individual molecule depend on the several factors.
The most important of which are :-
• Net charge.
• Charge ratio.
• Molecular shape.
• The temperature, porosity and viscosity of the matrix through which the
molecule migrate.
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3. GEL ELECTROPHORESIS
Gel electrophoresis is a separation technique in which a macromolecules such as DNA, RNA, Protein, etc.
can be separated based on their Charge, Size , Molecular weight Or other properties in the presence of an
electric field using gel as supporting medium.
The gels, however are porous and the size of the pores relative to that of the molecule determines whether the
molecule will enter the pore or bypass it. The separation thus not only depends on the charge on the
molecule, but also on its size. The resolution of sample is sharper and better in a gel than in other type of
medium.
Agarose gel is used as a supporting media for the separation of DNA, RNA or protein under the influence of
electric charge.
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4. PRINCIPLE
• The principle involved in gel electrophoresis is the separation of charged
particles from the sample applied on gel based on their size, charge, molecular
weight or any other property with the help of electric field.
• In this process, larger molecules have difficulty in moving through the pore
size of the supporting media, whereas the smaller molecules has more
mobility through it.
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5. TYPES OF GEL
Agarose Gel For separating larger nuclei acids
Polyacrylamide
Gel
•For separating smaller nuclei acids.
SDS-PAGE For denaturing the proteins.
STARCH Non- denatured proteins can be separated according to
charge and size.
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6. ELECTROPHORESIS BUFFER
• Composition and ionic strength of electrophoresis buffer is most important factor for the separation of nucleic acids
(DNA or RNA).
• TAE- (Tris-acetate-EDTA) :- It has lower buffering capacity and generally used to separate larger nucleic acid fragments.
• TBE- (Tris-borate-EDTA) :- it has high buffering capacity and higher ionic strength and generally used for separation of
low molecular weight compound.
• Loading buffer :- Nucleic acid is before loading onto a gel is first mixed with the gel loading buffer, which usually
consists of
• Salts :- It creates environment with favorable ionic strength and pH of the sample ex :-Tris-HCl
• Metal chelator :- It prevents nucleases to degrade the nucleic acids such as EDTA.
• Loading dyes :- It provides color for tracking and easy monitoring of samples such as bromophenol blue , xylene cyanol.
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7. REQUIREMENTS
Electrophoresis apparatus
• Casting tray of glass or plastic.
• Comb contains various number of teeth. (For formation of well)
Buffer
• It provides ions that carry current and maintain pH at constant
• value.
Power supply
• The electrodes are connected to their respective terminals of chamber and power supply
with controlled rate of current for best resolution, 5 volts per centimeter to the gel.
Supporting media (gel)
• Starch, Agar cellulose acetate, polyacrylamide.
• The choice of supporting media depends on type of molecules to be separated
Detection and Quantification
• Stains :-
• Protein staining.
• Ethidium bromide staining.
• Blotting :-
• Southern blotting (For DNA)
• Northern blotting (For RNA)
• Western blotting (For Protein)
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8. INSTRUMENTS
AND WORKING
.
GEL ELECTROPHORESIS
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Firstly, the sample of DNA is cutted into fragments by restriction endonucleases.
The DNA fragments are negatively charged and can be separated by forcing
them to move towards anode under electric field through medium or matrix.
The commonly used matrix is agarose, which is a natural linear polymer of D-
galactose and L- galactose which is extracted from the seaweeds
The DNA fragments are separated out according to their size because of the
sieving property of agarose gel. Hence, smaller the fragments further it will
move
The separated DNA fragments can be visualized by staining the DNA with
ethidium bromide followed by exposure to UV radiation
The DNA fragments are seen as orange color bands.
The separated bands of DNA are cutted out and extracted from the gel piece and this
step is called as elution.
9. APPLICATION
Used for the estimation of molecular weight of protein and nucleic acids.
Determination of subunits is structure of proteins.
Purification of isolated proteins.
Monitoring changes of protein content in body fluids.
Identifying disulfide bonds between proteins.
Quantifying proteins.
Blotting applications.
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10. ADVANTAGES
• High resolving power and Sharp Jones are obtained.
• Chemically inert.
• Available in wide range of pore size.
• Never bind to proteins.
• Stable over wide range of pH, temperature and ionic strength
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11. FACTORS AFFECTING SEPARATION
SAMPLE
• The charges or mass ratio of the sample indicates it’s electrophoretic mobility.
• The mass depends on molecular size and shape
• Charge :- Higher the charge, greater is the electrophoretic mobility.
• Size :- Bigger the molecules, greater the frictional and electrostatic forces exerted on it
by the medium therefore, larger particles have smaller electrophoretic mobility as
compared to smaller particles.
• Shape :- Rounded contour produce lesser friction and electrostatic force .
MEDIUM
• An inert supporting medium is generally selected for electrophoresis. This medium can be
exert adsorption or molecular saving effect on the particles, thus influencing the migration
rate.
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12. PH
• The direction and extent of migration of ampholytes , depend on the buffer pH.
• Buffer having pH between 1to11 are used for achieving the desired separation
IONIC
STRENGTH
• If the ionic strength of buffer increases, it indicates that the buffer ions will carry a larger share of
current, while the sample ions will carry a small part. This slows down the migration of sample
components.
• If the strength of buffer decreases, it indicates that the sample ions will carry a larger size of the
current, thus resulting in faster separation.
BUFFER
• The buffer maintains the pH of supporting medium and also affects the electrophoretic mobility of
the sample.
• The commonly used buffer are phosphate, acetate, Barberton, etc. The choice of buffer depends on
the type of sample to be separated by electrophoresis.
• Buffer affected the electrophoretic mobility if it binds to the sample component being separated.
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