I recently performed a Western blot analysis using a sodium potassium ATPase antibody on HeLa cells. The results showed a band below 100 kD and a cluster between 100 and 150 kD. However, literature and validation images typically report a band just above 100 kD for this protein.
Can anyone provide insights into what might be causing these discrepancies? Any suggestions for troubleshooting or optimizing the experimental conditions would be greatly appreciated!
Based on your description, the possible causes are speculated to be incomplete lysis, or the failure of the reducing agent in the loading buffer (the loading buffer may become ineffective if stored at room temperature or subjected to multiple freeze-thaw cycles).
You can try the following steps:
1. Ensure sufficient lysis: Use SDS or RIPA lysis buffer and perform lysis at 4°C for 30 minutes.
2. Large proteins tend to form aggregates and precipitates in the gel. Therefore, when transferring the proteins (membrane transfer), add SDS to the electrotransfer buffer to a final concentration of 0.1% to avoid this phenomenon. Methanol easily causes SDS to dissociate from proteins, so the concentration of methanol in the electrotransfer buffer should be reduced to 10% or lower to prevent protein precipitation. Additionally, reducing the proportion of methanol in the electrotransfer buffer can promote the swelling of the gel, facilitating the transfer of large proteins out of the gel.
3. Use a 0.45 μm PVDF membrane.
The answer to this question is provided by MedChemExpress Technical Support.
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