Conditions of gel
Denaturation:
The gels that are
denaturing can be used in the conditions which destroy the natural structure. The
movement of each molecule depends on the size and charge of the molecules. The
molecules especially proteins contain the primary, secondary and tertiary
structure are destroyed by using this type of gel and just primary structure
remains.
If we want to denature
nucleic acids than we can use urea but if we want to denature proteins SDS is
used. If we want to fully destroy proteins than their disulfide bonds are
broken as the disulfide bonds stabilize the different levels of structure.
In order to estimate
the RNA the denaturing conditions are essential. RNA forms more interactions as
compared to DNA. In order to disrupt the RNA we can add different agents like
urea.
Native:
The other type of gel
conditions is native conditions in which there are no denaturing conditions and
the native structure is maintained. We can analyze the four levels of structure
by using this type of gel conditions. In case of biological samples the
detergents are used to damage the membranes.
In this case it is difficult
to determine that how the size and charge of the molecules affect the mobility.
In this case the charged detergent is not used. The molecules are separated on
the basis of their size and charge and also on the basis of cross sectional
area and they face different type of electric forces.
As in this case the
native structure is not destroyed so we can’t observe proteins just by using
reagents but also enzymes are used for the staining purpose. During the
purification of protein in order to check the presence of enzyme we can do this
by checking the activity of enzymes.
Buffers:
In the process of gel
electrophoresis we use buffers for charge and in order to maintain the ph of
the solution. The buffers provide large amount of ions in order to provide current.
Water or benzene cannot be used for this purpose as they don’t have enough ions
to provide current.21
There are many buffers
that can be used for this purpose and the buffers that are most commonly used
are EDTA and TAE. Those buffers which have longer buffer life are advantageous.
The use of borate buffer causes problems as it has the ability of
polymerization.
The buffer TAE has
lowest capacity of buffer than other buffers. In the presence of this buffer we
can observe large fragments of DNA. It takes time but provides a better
product. Another buffer LB is ineffective as it cannot resolve those fragments
which has size larger than 5Kb.
Although it is
ineffective buffer but it can provide high voltage. It takes less time to
complete the process. A discontinuous buffer system is used in the case of
SDS-PAGE. If the process of electrophoresis is carried out in the presence of
discontinuous buffer system than at the start of the process an ion gradient
will be developed.
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