The topic for today is 2-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE), a technique to separate different proteins in a given sample. It is used to study the entire proteome of a cell. Today we shall understand the principle and the entire technique in brief. The next few following blogs we shall see each step in detail.
Before starting with the 2-D PAGE, it is really necessary to know the about the SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Isoelectric focussing electrophoresis.
– SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE):
To begin with, Electrophoresis literally means the movement of charged particles in a fluid or gel under the influence of the electric field. Electrophoresis was initially carried out in free solutions, later the solid supports were used, and with further development, now a days it is carried out in the gel networks like agarose and polyacrylamide gel.
Polyacrylamide gels are formed by the polymerization of acrylamide with small amount of N, N’ methylene bisacrylamide in presence of TEMED and APS. Polyacrylamide gels are more stronger and more suitable for the separation of proteins and nucleic acids according to their size or size/charge ratio. The electrophoresis carried out in Polyacrylamide gel is termed as Polyacrylamide Gel Electrophoresis (PAGE).
Many proteins are folded into compact structures, held together by a variety of non-covalent, ionic interactions such as hydrogen bonding and salt bridges. Such ionic bonds can be disrupted to denature the proteins and then the denatured proteins are separated. This step gives important structural information about the proteins.
Sodium dodecyl sulphate (SDS), an anionic detergent, can disrupt the folded structure of the protein. SDS consists of a 12-Carbon chain and a polar sulphated head. It binds non-covalently to proteins, with a stoichiometry of around one SDS molecule per two amino acids. SDS causes proteins to denature and dissassociate from each other (excluding covalent cross-linking). It also confers a negative charge, hence in the presence of SDS, the intrinsic charge of a protein is masked. During SDS PAGE, all proteins migrate toward the anode (the positively charged electrode). SDS-treated proteins have very similar charge-to-mass ratios and shapes. Therefore during PAGE, the rate of migration of SDS-treated proteins are determined by its molecular weight solely. This technique is called SDS- PAGE.
The isoelectric point (pI) is the specific pH at which the net charge of the protein is zero ( zwitterion), that is there are equal number of positive and negative charges, and not that there are no charges. Proteins are positively charged (cation) at pH values below their pI and negatively charged (anion) at pH values above their pI. If the pH of the electrophoretic medium is identical to the pI of a protein, the protein has a net charge of zero and does not migrate toward either electrode, that the pull from both the electrodes are of equal strength. Hence if a mixture of proteins is run in a gradient of pH, the individual proteins will cease to move whenever the pH is identical to their pI, thus getting separated on the basis of their pI. This technique of separating proteins is called Isoelectric focussing (IEF).
Now with this information, we can put together the 2-D PAGE….
2-D PAGE or 2-DE is a technique which can give enhanced resolution of complex protein mixtures. It is a powerful tool for researchers in the field of proteomics, to study the structure, interactions, and biological function of the proteins expressed by the genome (proteome) of an organism. It was first reported by P.H. O’Farrell in 1975. In 2DE the protein mixture is first separated by Isoelectric focussing and then the separated proteins (each band) are further separated by SDS- PAGE.
As previously mentioned, in the first dimension, that is IEF, the proteins are separated according to their isoelectric point (pI). IEF of the protein sample is performed on a thin strip. After the completion of IEF, the strip is run perpendicular to the first run along the SDS- PAGE gel, the second dimension, wherein the proteins with similar pI (grouped together) are further separated on the basis on their molecular weight on SDS-PAGE gel (see fig 2).
Hence all the protein are separated twice, with second run perpendicular to the first one, as shown in figure. The proteins in the SDS-PAGE gel shown in same color (in fig 2) have same/similar pI.
Every spot on the gel correspond to a single protein type, and thousand such protein spots will be obtained. The increase or decrease in intensity of a spot representing a specific protein may be monitored as a function of cell function.
However with thousands of spots, the analysis is complex, time-consuming and difficult. But with recent developments, the data can be analysed using modern computer software and online protein databases.
Experimentally the entire process is carried out in different steps:
- Sample preparation
- IPG strip rehydration
- IPG strip equilibration
The proteins separated by 2-DE can be further analyzed by mass spectrometer and partial protein sequence data can be obtained.
In the next few posts, we shall see the details of each step in the 2DE.
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