What are the possible reasons for the protein molecular weight detected by SDS-PAGE being greater than expected?

When you find that the molecular weight of a protein is greater than expected in SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) analysis, there could be several reasons:

1. Protein Aggregation:

In some cases, proteins may form dimers or larger polymers. This could be due to the nature of the protein itself or conditions during sample processing (e.g., pH, temperature, ionic strength). If proteins aggregate, their migration rate in SDS-PAGE will be slower than that of individual protein molecules, leading to a detected molecular weight greater than expected.

2. Post-translational Modifications:

Proteins may undergo various chemical modifications after translation, such as phosphorylation, glycosylation, ubiquitination, etc. These modifications can increase the molecular weight of the protein, resulting in a higher than expected molecular weight detected by SDS-PAGE.

3. Protein Shape:

SDS-PAGE separates proteins based on molecular weight, but the shape of the protein can also affect its migration in the gel. Some non-spherical proteins may migrate slower than their actual molecular weight suggests, leading to a detected molecular weight greater than expected.

4. Sample Preparation Issues:

If the sample is not fully dissolved in SDS or not completely reduced by β-mercaptoethanol or DTT (dithiothreitol), proteins may exist as polymers, resulting in a higher than expected molecular weight detected by SDS-PAGE.

5. Gel Electrophoresis Conditions:

If the electrophoresis conditions (e.g., voltage, electrophoresis time, gel concentration) are not set correctly, they may affect the migration of proteins in the gel, resulting in a higher than expected molecular weight detected by SDS-PAGE.

To address this issue, you can try optimizing sample processing and electrophoresis conditions or use other techniques (e.g., mass spectrometry, Western blotting) to further verify your results.

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