Western Blot: Why Did This Situation Occur Where the Protein Ran onto the Marker
When proteins run onto the marker during a Western blot experiment, several factors may contribute to this phenomenon:
1. Excessive Sample Loading
Loading an excessive amount of sample can cause proteins to migrate beyond the resolving capacity of the gel, resulting in some proteins running onto the marker. This typically occurs when the protein concentration is too high or when the sample volume is not accurately controlled during loading.
Solution:
Determine the loading volume based on both the protein concentration and the desired resolution range. If the protein concentration is excessively high, dilute the sample appropriately before loading.
2. Protein Aggregation
Certain proteins may aggregate during electrophoresis, leading to aberrant migration and accumulation on the marker. This behavior is often associated with specific protein properties or unsuitable experimental conditions.
Solution:
Incorporate non-ionic detergents such as SDS (sodium dodecyl sulfate) into the electrophoresis buffer to minimize aggregation. Additionally, optimizing electrophoresis parameters—such as voltage and run time—can further reduce aggregation.
3. Abnormal Protein Migration
Irregularities during electrophoresis, such as uneven electric fields or overextended running times, may result in uneven protein migration, causing some proteins to run onto the marker.
Solution:
Ensure consistent electrophoresis conditions by using freshly prepared buffers and appropriately configuring the electric field strength and run duration. Also, inspect the electrophoresis system for potential issues, such as improper electrode placement or electrical resistance between electrodes.
4. Protein Degradation
Degraded proteins may migrate unpredictably during electrophoresis, potentially running onto the marker.
Solution:
To preserve protein integrity during sample preparation, minimize degradation by working at low temperatures and including protease inhibitors. These measures help maintain sample stability and prevent degradation-related migration artifacts.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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