Separation of a protein mixture using SDS-PAGE gels is given as a function of its size, which allows the approximate molecular weight of a protein to be calculated.
Despite the fact that certain proteins can suffer up to 10% deviation from their real molecular weight when using this method (especially for those proteins with post-translational modifications), SDS-PAGE continues to be one of the most widely used techniques due to its simplicity and simplicity.
In this post we summarize how to calculate the molecular weight of a protein using SDS-PAGE in 5 easy steps .
5 STEPS TO CALCULATE THE MOLECULAR WEIGHT OF A PROTEIN
Calculating the molecular weight of a protein using SDS-PAGE is as simple as comparing the electrophoretic mobility of the problem protein with that of a series of reference proteins whose molecular weight we know. To do this, we just have to follow these 5 simple steps
1.- SEPARATE THE PROTEINS
First, the sample containing the problem protein is run on the same gel together with a series of standards or reference proteins whose molecular weight is known.
2.- PROCESS THE GEL WITH THE CORRESPONDING STAINING
Once the proteins are separated, the gel is processed with the relevant staining to be able to visualize the bands corresponding to each one of them.
3.- CALCULATE THE RELATIVE MIGRATION DISTANCE
With the defined bands, we proceed to calculate the relative migration distance (Rf), or relative mobility, both of the problem protein and of each of the reference proteins, according to the following equation:
The front of the gel refers to the position therein of the band corresponding to a maximum mobility reference compound, such as bromophenol blue.
The migration distance is measured in centimeters, and can be calculated manually using a ruler, or through specific software.
4.- GENERATE A GRAPH
The logarithm values of the molecular weight corresponding to each of the standard proteins are represented in a graph against the values of the relative migration distance (Rf) corresponding to each of them. This will give us a straight line (as long as the proteins are totally denatured, and the percentage of gel is in line with the molecular weight range of said proteins).
5.- EXTRAPOLATE THE VALUE OF THE PROBLEM PROTEIN
Finally, the value of the relative migration distance (Rf) of the problem protein is extrapolated in the graph to obtain its estimated molecular weight. Et voilà!