Q.5. (c) Explain Electrophoresis, its principle and factors governing it
Ans.5.(c) Electrophoresis: is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology.
Electrophoresis is defined as the migration of charged ions in an electric field. In metal conductors, electric current is carried by the movement of electrons, largely along the surface of the metal. In solutions, the electric current flows between electrodes and is carried by ions. The ions that migrate towards the anode, because of their anodic migration, are called “anions”. The ions which will migrate to the cathode are called “cations”.
Principle: When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.
An ion placed in such an electric field will experience a force:
Where,
F = electrophoretic force
K = a constant
q = net charge on the protein (atomic charges/protein molecule)
This force will cause the protein to accelerate towards either the cathode or the anode, depending on the sign of its charge. Of course there are other forces such as frictional force when ions move in the electric field. The influence of them cannot be understood easily by a formula, so we omit it.
Electrophoresis exploits the fact that different ions have different mobility in an electric field and so can be separated by this way.
Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value.
The gel itself is composed of either agarose or polyacrylamide, each of which has attributes suitable to particular tasks.
Factors influencing Electrophoresis:
Movement of proteins depends on various aspects. Within the gel the molecules must pass through as they are moving from one pole to another. The smaller molecules can weave in and out of the matrix of the gel with more ease, compared with larger molecules. As a general rule, the molecules move rapid if it has more net charge, has a shape of ball and shorter diameter
1) The buffer pH
It will influence the direction and rapid of the protein migration.
Movement of proteins depends on various aspects; one of them is the charges on the proteins. Proteins are sequence of amino acids that can be ionized depend on their acid or basic character. The protein’s net electric charge is the sum of the electric charges found on the surface of the molecule as a function of the environment.
The rate of migration will depend on the strength of their net surface charges: The protein that carries more +ve charges will move towards the cathode at a faster rate. On the contrary, the protein that carries more -ve charges will move towards the anode at a faster rate. In this regard, proteins can be separated based on their electric charges.
Depending on the pH of the buffer, proteins in a sample will carry different charges. At the pI (isoelectric point) of a specific protein, the protein molecule carries no net charge and does not migrate in an electric field. At pH above the pI, the protein has a net negative charge and migrates towards the anode. At pH below the pI the protein obtains a net positive charge on its surface and migrates towards the cathode.
2) The buffer ionic strength
It influences the proportion of the current carried by the proteins
At low ionic strength the proteins will carry a relatively large proportion of the current and so will have a relatively fast migration. At high ionic strength, most of the current will be carried by the buffer ions and so the proteins will migrate relatively slowly. An analogy might be useful in visualizing this effect of ionic strength. Imagine a bank where there are two counters – one for deposits the anode) and one for withdrawals (= the cathode), with electrons being the money. The ions may be considered as customers waiting to be served at either counter, which one can visualize as being at opposite ends of the banking hall.
In electrophoresis, therefore, a low ionic strength is preferred as it increases the rate of migration of proteins. A low ionic strength is also preferred as it gives a lower heat generation. Assuming a constant voltage, if the ionic strength is increased, the electrical resistance decreases but the current will increase. A high ionic strength buffer will therefore lead to greater heat generation, and so a low ionic strength is preferred.
3) The voltage gradient
The rate of migration will depend on the voltage gradient: There is more voltage gradient in the electric field, protein will move towards the anode (or the cathode) at a faster rate.
4) Electo-osmosis
Liquid’s relative move upon solid medium in an electric field is called electo-osmosis. In applied electric field, electo-osmosis distorts the sample stream and limits the separation. For example, Paper electrophoresis has poor resolution because of electo-osmosis. The surface of paper has -e, so the buffer has +e derived from hydrogen ions because of electrostatic induction.
Then +e drive buffer to cathode in electric field,these flows distort the electrophoretic migration of sample by causing a varying residence time. Thus, sample will move more or less than normal.
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