How to determine the molecular weight of a protein?
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Secondary SchoolBiology 10+5 pts
How you determine molecular weight of protein by using SDS-PAGE?
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GopikaNokhwal
GopikaNokhwal Virtuoso
Molecular Weight DeterminationSo, how does SDS-PAGE work in determining the size or molecular weight of unknown proteins? Here's everything you need to know.
SDS-PAGE for Molecular Weight Determination: An Overview of the Process
To determine the molecular weight of an unknown protein, you should separate the sample on the same gel with a set of molecular weight standards. After running the standards and the unknown protein sample, the gel is processed with the desired stain and then de-stained for about 12 to 14 hours to visualize the protein bands.
After running the gel, you should then determine the relative migration distance (Rf) of the protein standards and the unknown protein. The migration distance can be determined using the following equation:
Rf = migration distance of the protein
Migration distance of the dye front
Note: You can use a ruler to measure the migration distance (in centimeters) from the top of the gel to every major band in the gel. Alternatively, an appropriate software may also be used to determine the Rf values of the resulting bands.
Based on the values obtained for the bands in the standard, the logarithm of the molecular weight of an SDS-denatured polypeptide and its relative migration distance (Rf) is plotted into a graph. Please take note that you will generate a linear plot for most proteins if your samples are fully denatured and the gel percentage is appropriate for the molecular weight range of the sample. If you get a sigmoidal curve, it means that the sieving effect of your matrix is either too large that it restricts the penetration of the molecules into the gel or is nearly negligible that it allows protein molecules to migrate almost at their free mobility.
Interpolating the value from this graph will then give you the molecular weight of the unknown protein band. Please note that the accuracy of this method in determining the molecular weight of an unknown protein typically ranges from 5% to 10%. The presence of polypeptides such as glycol- and lipoproteins usually leads to erroneous results since they are not fully coated with SDS and thus, would not behave as expected.
So for the example pictured, the unknown protein has an Rf of 0.7084. Using the equation for the linear plot we can calculate the Log (MW). (-2.0742 x 0.7084) +2.8 = 1.3305. So the inverse log is 10^1.3305= 21.4kDa for the molecular weight of the unknown protein.
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Lazurite
Lazurite Ambitious
The SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) method. Although there are some other methods (i.e. analytical ultracentrifugation and light scattering) for calculating the size or molecular weight (MW) of an unknown protein, they are not commonly used for this purpose since they use large amounts of highly purified proteins and require costly equipment.
To determine the molecular weight of an unknown protein, you should separate the sample on the same gel with a set of molecular weight standards. After running the standards and the unknown protein sample, the gel is processed with the desired stain and then de-stained for about 12 to 14 hours to visualize the protein bands.
After running the gel, you should then determine the relative migration distance (Rf) of the protein standards and the unknown protein. The migration distance can be determined using the following equation:
Rf = migration distance of the protein
Migration distance of the dye front