Why Do Large Molecular Weight Protein Bands Appear Wavy in Western Blot
In proteomics research, Western Blot is a common method for measuring protein expression levels. A common issue observed in Western Blot experiments is the wavy appearance of large molecular weight protein bands (also known as the "smile effect"). This can be caused by several factors:
1. Electrophoresis Conditions
(1) Voltage Settings: If the electrophoresis voltage is too high, it may cause proteins to migrate too quickly, leading to wavy bands.
(2) Electrophoresis Time: Both overly long and short electrophoresis times can affect protein migration.
2. Gel Issues
(1) Gel Concentration: An inappropriate gel concentration for large molecular weight proteins may lead to uneven separation.
(2) Gel Preparation or Storage: Errors in gel preparation or long-term storage may affect its performance.
3. Buffer Issues
(1) Buffer Composition: Incorrect pH or ionic strength of the buffer may impact protein migration.
(2) Buffer Temperature: Overheating of the buffer can lead to local gel deformation, affecting protein migration.
4. Sample Issues
(1) Sample Loading Amount: Too much or too little sample loading can affect band shape.
(2) Sample Treatment: Inadequate denaturation or reduction of samples may influence their migration through the gel.
Solutions
1. Optimize electrophoresis conditions by adjusting voltage and time to ensure proper protein migration.
2. Select the appropriate gel concentration based on the size of the protein.
3. Ensure proper buffer composition and temperature, using fresh buffer and preventing overheating during electrophoresis.
4. Optimize sample preparation and loading to ensure full denaturation and reduction, and load an appropriate amount of sample.
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