What Are the Possible Reasons for the Molecular Weight of Proteins Detected by SDS-PAGE Being Higher Than Expected
When analyzing proteins using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), the observed molecular weight may exceed the expected value due to several factors:
1. Protein Aggregation
Under certain conditions, proteins can form dimers or larger aggregates. This may result from the intrinsic biochemical properties of the protein or from specific conditions during sample preparation, such as variations in pH, temperature, and ionic strength. If aggregation occurs, the migration rate of these proteins in SDS-PAGE will be slower than that of monomeric proteins, leading to an apparently higher molecular weight.
2. Post-Translational Modifications
Proteins often undergo various post-translational modifications, such as phosphorylation, glycosylation, and ubiquitination. These modifications introduce additional molecular mass, which can lead to an apparent increase in molecular weight when analyzed by SDS-PAGE.
3. Protein Conformation
Although SDS-PAGE primarily separates proteins based on their molecular weight, their three-dimensional structure can also affect migration. Certain non-globular proteins may exhibit slower migration compared to what is predicted based on their molecular weight alone, leading to an apparently higher molecular weight.
4. Incomplete Sample Preparation
If the protein sample is not fully denatured by SDS or incompletely reduced by β-mercaptoethanol or DTT (dithiothreitol), residual disulfide bonds may cause protein aggregation. As a result, SDS-PAGE may detect an artificially elevated molecular weight.
5. Suboptimal Electrophoresis Conditions
Improper electrophoresis parameters, such as voltage settings, electrophoresis duration, and gel concentration, can influence protein migration behavior, potentially leading to an observed molecular weight that deviates from the expected value.
To resolve these discrepancies, optimizing sample preparation protocols and electrophoresis conditions is recommended. Additionally, alternative techniques, such as mass spectrometry and Western blotting, can be employed for further validation of the results.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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