Determination of Oligomeric status of the protein- The polyacrylamide gel electrophoresis can be use to determine the oligomeric status of the protein. A protein sample can be run under the denaturating as well as in the native conditions in two separate gel. The protein of the known molecular weight runs on both gels and a Rf value is calculated for the standard proteins as described. A calibration curve from native and denaturating gel is used to determine the molecular weight (native and denaturating) of the protein. The oligomeric status of the protein is calculated from the formula given below:

Studying protein folding/unfolding- The urea disrupts electrostatic and hydrophobic interaction between amino acid residues of protein to induce unfolding of the 3-D conformation of the protein. It has been extensively used to study the protein unfolding and to identify different structural intermediates in the folding pathways. In a typical unfolding experiment, protein is exposed to the different concentration of urea and then the structural changes in protein can be monitored by the spectroscopic or gel filitration techniques. Unfolding of protein causes an increase in the hydrodynamic volume of the protein and it results in slower mobility in polyacryalmide gels. In the urea PAGE, a polyacrylamide gel is prepared with a horizontal gradient of urea (0-8M). The same protein sample is loaded in different lanes and it is allowed to run vertically, perpendicular to the urea gradient. As sample runs in different lanes, it gets exposed to different concentration of urea and consequently at a particular urea concentration the protein is unfolded with an increase in hydrodynamic volume. The unfolded protein sample will migrate slower due to increase in friction forces and it gives a unique protein band pattern to provide qualitative or semi-quantitative information about the protein folding intermediates (Figure 23.2). The information from the gradient urea PAGE needs further verification from other analytical techniques. In addition to protein folding, urea PAGE can also be used to analyze the protein complexes as well as covalent heterogenecity of the protein.