Possible Causes for Target Bands Merging into One in WB, While Internal Control is Normal?
If your Western Blot (WB) target bands merge into a single band, but the internal control is normal, the possible causes could include:
Sample Overloading
If too much of the target protein sample is loaded, the bands may merge into one. Make sure to load an appropriate amount of sample to avoid overloading.
High Antibody Concentration or Affinity
If the primary or secondary antibody has too high an affinity or concentration, it may lead to over-staining and cause the bands to merge.
Excessive Migration Time
If the migration time is too long, it can cause the bands to spread out and appear as a single band. Ensure that the migration time is appropriately controlled.
Uneven Distribution of Protein Concentration
If the target protein concentration in the sample is uneven, some areas may have higher protein concentrations, causing continuous bands to form. Ensure better mixing of the protein during the sample preparation stage.
Insufficient Blocking or Over-Washing
If the blocking step is inadequate or the washing step is too thorough, it could lead to non-specific binding or overly strong signals.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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