One can calculate the molecular weight of protein by using SDS-PAGE (sodium dodecyl polyacrylamide gel electrophoresis). Other methods like analytical ultracentrifuge and light scattering can also be used for this purpose. But SDS-PAGE is the preferred method as it is cost-effective and requires less amount of purified sample. In this method, the unknown protein is separated along with the molecular weight standard. Smaller proteins move faster and thus proteins are separated according to their size.
To calculate the molecular weight we first find out the relative migration distance of protein by using the following formula:
Relative migration distance (Rf)= migration distance of the protein/migration distance of the dye front
Now a graph is plotted between log molecular weight and relative migration distance of the standard protein. The molecular weight of the unknown protein is calculated from this standard linear graph by using the linear plot equation.
In this way one can calculate molecular weight of the protein without using bioinformatics tool.