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Methods in Biology
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Pulsed
field gel electrophoresis - is a technique used for the separation
of large DNA molecules by applying to a gel matrix an electric
field that periodically changes direction. PAGE is used to
separate proteins or nucleic acids, according to their
electrophoretic mobility. Electrophoretic mobility is a function
of the length, conformation and charge of the molecule.
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Chromatography
- is a method of separating the constituents based on one or more
of its chemical properties such as charge, polarity etc.
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Centrifugation
- is a mechanical process which involves the use of the
centrifugal force to separate particles from a solution
according to their size, shape, density, medium viscosity and
rotor speed.
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Large DNA
molecules are separated using - pulsed field gel electrophoresis.
Microscopy will only aid in visualization.
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Methods in Biology
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Isoelectric focusing (IEF), also known as electrofocusing, is a
technique for separating different molecules by differences in
their isoelectric point (PI). pl is the pH at which a molecule
carries no net electrical charge or is electrically neutral. It is
calculated by taking the average of different pKa values which
represent various acidic (negative) and basic (positive) groups.
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High salt concentration will stabilize
protein and DNA by reducing electrostatic forces and making
structure more compact. Volume would reduce and migration will
come rapid. Protein Would migrate as a homotetramer in presence of
hydrophobic interactions because salt will increase them. Protein
would migrate as a monomer in Presence of electrostatic
interactions because salt will reduce them. DNA will not become
single stranded. Correct answer is 4: 3 different sized proteins
may have same pl corresponding to the same band in IEF but
different band in SDS PAGE. It can also be inferred that a single
protein may be composed of 3 subunits and on subjecting to SDS
treatment, it gives 3 bands of different molecular weights,
40kDa + 20 kDa = 60 kDa (dimer) 60 kDa + 60
kDa = 120 kDa (tetramer)
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