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.