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Gel Electrophoresis Project

Gel electrophoresis is a technique used to separate biomolecules like DNA and proteins based on their size and charge. It works by applying an electric field to a gel which causes the molecules to migrate at different rates depending on factors like their mass and conformation. Agarose gels are commonly used to separate DNA fragments while acrylamide gels can separate smaller DNA and denature proteins so they separate based on mass. The technique allows determination of properties like molecular weight and purity of samples.

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60 views3 pages

Gel Electrophoresis Project

Gel electrophoresis is a technique used to separate biomolecules like DNA and proteins based on their size and charge. It works by applying an electric field to a gel which causes the molecules to migrate at different rates depending on factors like their mass and conformation. Agarose gels are commonly used to separate DNA fragments while acrylamide gels can separate smaller DNA and denature proteins so they separate based on mass. The technique allows determination of properties like molecular weight and purity of samples.

Uploaded by

vidhya suresh
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© © All Rights Reserved
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Gel Electrophoresis

Gel electrophoresis is a simple technique, introduced in the early 1970 s, which truly
revolutionized the biophysical studies of DNA and RNA and, subsequently, the whole field of
molecular biology. All developments in this field in the past 40 years are connected, directly or
indirectly, with the gel electrophoresis method. Gel electrophoresis has pushed aside
ultracentrifugation as the method to separate nucleic acids.
Gel electrophoresis differs from electrophoresis in solution only in the nature of the medium in
which molecules are separated in the electric field. In case of gel electrophoresis the medium is
the gel, a polymer network. The most popular in the field of nucleic acids are gels made of
polyacrylamide or agarose. Originally, the great advantage of gels in the separation of nucleic
acids was discovered purely empirically. The understanding came later after some ideas of P-J
De Grennes were borrowed from polymer physics, namely the notion of reptation of polymer
molecules.
Gel electrophoresis is a well-established molecular biology technique
typically used for the separation of biomolecules such as DNA and
proteins [203,204]. It is based on the electrokinetic phenomena in
which charged particles or molecules migrate relative to a fluid in the
presence of an electric field [205]. The differential electrophoretic
mobility of molecules in a gel matrix arising from the relative
disparities in size, shape, and charge allows for the distinct separation
of molecules in solution.
In addition to biomolecules, nanoparticles of various shapes and sizes
can also be differentiated based on the principles of gel
electrophoresis as well [206]. This approach can also be extended to
the isolation and identification of DNA–nanoparticle conjugates
Gel electrophoresis is used to characterize one of the most basic properties - molecular
mass - of both polynucleotides and polypeptides. Gel electrophoresis can also be used
to determine: (1) the purity of these samples, (2) heterogeneity/extent of
degradation, and (3) subunit composition.

DNA

The most common gel electrophoresis materials for DNA molecules


is agarose and acrylamide.

DNA agarose gels

The electrophoretic migration rate of DNA through agarose gels is dependent


upon four main parameters:
1. The molecular size of the DNA. Molecules of linear duplex DNA travel through
agarose gels at a rate which is inversely proportional to the log of their molecular
weight.

Mr∝1/log(Mw)(10.3.1.1)

3. The conformation of the DNA.

 closed circular DNA (form-I) - typically supercoiled


 nicked circular (form-II)
 linear DNA (form-III)

These different forms of the same DNA migrate at different rates through an agarose


gel. Almost always the linear form (form-III) migrates at the slowest rate of the
three forms and supercoiled DNA (form-I) usually migrates the fastest.

4. The applied voltage.

 Typical value for running an agarose gel is 5 volts per cm (length of gel)
Finally, the DNA being an acidic molecule, migrates towards
the positively charged electrode (cathode).

DNA acrylamide Gels

Acrylamide gels are useful for separation of small DNA fragments typically


oligonucleotides <100 base pairs. These gels are usually of a low acrylamide
concentration (<=6%) and contain the non-ionic denaturing agent Urea (6M). The
denaturing agent prevents secondary structure formation in oligonucleotides and allows
a relatively accurate determination of molecular mass.

Gel Electrophoresis for Proteins

Gel electrophoresis of proteins almost exclusively utilizes polyacrylamide. The


acrylamide solution usually contains two components: acrylamide and bis acrylamide.
A typical value for the acrylamide:bis ratio is 19:1. The bis acrylamide is essentially
a cross-linking component of the acrylamide polymer. The total acrylamide
concentration in the gel affects the migration of proteins through the matrix (as with
the concentration of agarose).

Protein gels are usually performed under denaturing conditions in the presence of


the detergent sodium dodecyl sulfate (SDS). The proteins are denatured by heat in
the presence of SDS. The SDS binds, via hydrophobic interactions, to the proteins in an
amount approximately proportional to the size of the protein. Due to the charged
nature of the SDS molecule the proteins thus have a somewhat constant charge to
mass ratio and migrate through the gel at a rate proportional to their molecular mass,
The proteins migrate towards the anode.

 Both DNA and protein gel electrophoresis utilize molecular weight standards to
calibrate the size(s) of samples being analyzed
 DNA molecular weight standards will consist of a mixture of DNA fragments of
known sizes (molecular mass)
o One convenient DNA molecular weight standard is constructed by partial ligation
of a 100 base pair fragment of duplex DNA
 Partial ligation will result in formation of dimers (200 bp), trimers (300 bp) and
so on, as well as some amount of the original (100 bp) fragment
 This produces a DNA "ladder" after gel electrophoresis
o Another DNA fragment standard might be a known DNA sequence, such as the
plasmid pBR322, which has been digested with a four-cutter restriction
endonuclease (e.g. Alu I).
 This produces a variety of fragments with different sizes
 It is easily reproduced (just cut more pBR322)
 Protein molecular weight markers usually consist of a mixture of a half dozen or
so pure proteins with known molecular masses

https://chem.libretexts.org/Courses/California_Polytechnic_State_University_San_Luis_Obispo/
Survey_of_Biochemistry_and_Biotechnology/10%3A_Supplemental_Modules_(Biochemistry)/
10.03%3A_3._Biotechnology_1/10.3.01%3A_Gel_Electrophoresis

https://www.sciencedirect.com/topics/chemistry/gel-electrophoresis

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