What Causes Proteins to Migrate into the Marker Lane in Western Blot?
In a Western Blot experiment, proteins are separated during electrophoresis and transferred onto a membrane, where they bind to target proteins through specific antibodies, forming distinct protein bands. However, sometimes proteins may migrate out of the intended lane or multiple bands may appear during the process. Below are some possible causes of this issue:
1. Improper Electrophoresis Conditions
If the electrophoresis voltage or current is too high, it may cause the proteins to migrate too quickly and end up in the marker lane.
2. Inappropriate Gel Pore Size
If the pore size of the polyacrylamide gel is not suitable for the target protein size, the protein may not be properly retained by the gel and could migrate out of the lane.
3. Excessive Sample Loading
Overloading the sample may lead to uneven protein distribution on the gel, causing proteins to migrate into adjacent lanes or spread outside the intended lane.
4. Marker and Sample Contamination
Improper handling during sample loading may cause contamination between the marker and the sample, leading to interference.
To resolve this issue, the following steps may be helpful:
1. Check and adjust the electrophoresis voltage and running time.
2. Optimize the sample handling and loading amount.
3. Use gels with appropriate concentrations and pore sizes that are suitable for the target protein.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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