Why Is the Target Band Missing in WB When GAPDH and Positive Control Are Both Normal?
If such a situation occurs, several possible causes should be considered:
Low Expression Level of the Target Protein
The target protein may be expressed at a level too low to be detected. This could result from the intrinsic properties of the sample or from protein loss during sample preparation.
Insufficient Antibody Specificity or Affinity
The primary or secondary antibody used may not adequately recognize the target protein. It is essential to verify the specificity and activity of the antibodies. If necessary, try using alternative antibodies.
Protein Degradation or Loss During Sample Preparation
Protein degradation may occur if the sample is not properly lysed, if the lysis buffer is inappropriate, or if protease inhibitors are not added. Review the preparation process for potential steps that may contribute to protein loss or degradation.
Insufficient Sample Loading
If the quantity of protein loaded is too low, the target protein may fall below the detection threshold. Increasing the loading amount may help.
Inefficient Membrane Transfer
Incomplete transfer of proteins from the gel to the membrane during blotting may lead to a missing band. Ensure that both electrophoresis and transfer steps are performed correctly.
Low Sensitivity of the Detection Method
The detection method employed may not be sensitive enough to detect low-abundance proteins. Consider using more sensitive detection techniques.
To troubleshoot this issue, review and adjust each experimental step according to the above checklist. Careful attention to experimental details is critical. Consulting with colleagues or mentors can also provide valuable insights and help identify potential solutions.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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