Antibodies usually recognizes a small portion of the protein of interest, the epitope. The epitope domain is found inside the 3D conformation of the protein. To enable access of the antibody to this portion it is essential for the protein to be folded meaning to denature it (2). A SDS-PAGE will denature the protein, but denaturation is not the same as breaking S-S (Disulfide) bonds (3). A reducing gel will have DTT added to the sample before boiling, and will break any disulfide bonds in the protein. This results in a protein with a quaternary structure (multiple subunits) to produce equal number of bands as there are subunits in the protein (4). Non-reducing conditions, no DTT, will not break S-S bonds, thus maintaing the structure. Disulfide bridges can drastically change the tertiary structure of a protein and change the behaviour of the protein on a gel. Generally, disulfide bridges should make the protein more compact and make it run faster on a gel. If there is a 1 band in non-reducing and 2 bands in reducing sample, this means that the protein contains one or more disulfide bonds (5).
For a homodimer, its subunits are presumably connected by disulfide bond, if we use nonreducing SDS, the protein will appear as a single band on the gel. If we use reducing SDS, the disulfide bond is broken thus resulting in 2 identical dimers migrating across the gel. Since they are identical, their migration distance will be the same, and we still observe only 1 band on the gel (4). This greatly explains the size differences between bands in the reduced and unreduced lanes. The thickness of the band corresponds to the amount of protein present.
An antigen is any substance that the immune system can recognize as being foreign and which caused an immune response. Since antigens are too large to bind to any receptor, only specific segments that form the antigen bind with a specific antibody. Such segments are called epitopes. Antibodies can recognize linear or conformational epitopes. An antibody may recognize a linear epitope by its linear sequence of amino acids, or primary structure. Since these amino acids lie continuously in a line, they are referred to as linear epitopes (6). Or the antibody can also recognize amino acids that usually do not exist as straight chains but as folded whorls with complex loops. These surface amino acids are not continuous with each other, discontinuous amino acids that come together in three-dimensional conformation and interact with the receptor's paratope are called conformational epitopes (7).
The major envelope glycoprotein, E, of WNV is involved in viral attachment and entry via membrane fusion (2). From the western blot of Envelope protein (E), a single band of approximately was observed in lane 3, the unredu...
