Why Do Samples Form a Continuous Line During Protein Electrophoresis in WB
During WB experiments, the appearance of a continuous line of the sample on the membrane is due to the accumulation of proteins during electrophoresis separation and transfer, as well as the continuous distribution of proteins. This phenomenon may affect the interpretation of experimental results, so it is essential to optimize the sample loading and electrophoresis conditions to achieve optimal protein separation and transfer efficiency on both the gel and the membrane.
1. Accumulation Effect During Protein Electrophoresis Separation
During SDS-PAGE, protein samples are loaded into the gel wells and migrate under an electric field. Because proteins differ in size and charge, their migration speeds vary: larger proteins migrate more slowly, while smaller proteins migrate more quickly. This causes the proteins to separate into distinct bands within the gel.
2. Accumulation Effect During Protein Transfer
During the transfer of proteins from the gel to the membrane, typically performed by electrotransfer, proteins are carried from the gel onto the membrane by an electric field. As with electrophoresis, larger proteins transfer more slowly, and smaller proteins transfer more quickly, resulting in the formation of distinct bands on the membrane.
3. Continuous Distribution of Proteins
After separation into distinct bands within the gel, the proteins retain this band pattern during transfer to the membrane. If the proteins in a sample form a continuous band in the gel, this continuous band will appear as a continuous line on the membrane after transfer.
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