Why Does the Observed Protein Run Smaller Than Predicted in Western Blot
When a protein appears at a lower molecular weight than predicted in a Western blot (WB) experiment, several potential factors may be responsible:
1. Protein Modifications
Proteins within cells may undergo various post-translational modifications such as phosphorylation, methylation, or acetylation. These modifications can alter the protein’s charge, conformation, or molecular weight, thereby influencing its electrophoretic mobility. In some cases, such modifications may increase the protein’s migration speed in SDS-PAGE, causing it to appear at a lower apparent molecular weight than predicted.
2. Protein Conformation
The three-dimensional structure of a protein can also affect its mobility during electrophoresis. If the protein adopts a compact conformation, it may interact differently with SDS or experience altered migration through the gel matrix, resulting in an apparent molecular weight smaller than expected. Furthermore, improper SDS binding due to folding differences can affect the protein’s charge-to-mass ratio and influence migration.
3. Protein Degradation
Proteins may undergo degradation by cellular proteases. If degradation occurs during sample collection, lysis, or electrophoresis, smaller protein fragments may be generated, leading to bands that migrate faster and appear at lower molecular weights than predicted.
4. Experimental Conditions
Variations in experimental conditions—such as electrophoresis voltage, gel concentration, or running time—can significantly influence protein migration. Suboptimal conditions may cause artifacts in migration patterns, making proteins appear smaller than their expected molecular weights.
5. Molecular Weight Markers
The use of inappropriate or incompatible molecular weight markers may result in inaccurate estimation of the protein’s apparent molecular weight.
6. Alternative Protein Isoforms
Some genes express multiple isoforms, which may differ in size from the predicted canonical form. These isoforms can display distinct migration patterns, potentially explaining unexpected band positions.
To identify the underlying cause of such discrepancies, consider the following approaches:
1. Re-examine the sequence of your target protein and ensure that it corresponds to the epitope recognized by the antibody used in the Western blot.
2. Perform mass spectrometry analysis to confirm that the observed band corresponds to the protein of interest.
3. Optimize your sample preparation protocols to preserve protein integrity and prevent degradation.
4. Use a range of molecular weight markers to improve the accuracy of apparent molecular weight estimation.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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