Semi-Dry or Tank Electrotransfer Troubleshooting
Electrotransfer refers to the standard procedure for transferring proteins from a polyacrylamide gel (SDS-PAGE) onto an Immobilon® PVDF transfer membrane. The two commonly used electrotransfer techniques are tank transfer and semi-dry transfer. Both are based on the same principles and differ only in the mechanical devices used to hold the gel/membrane stack and applications of the electrical field.
Click on the Semi-dry or Tank Electrotransfer symptoms to read about the possible causes and remedies:
- Band Smeared/Distorted
- High Background in Fluorescent Detection
- No Signal
- Poor Transfer of Small Molecular Weight Proteins
- Poor Transfer of Large Molecular Weight Proteins (~ >80 kDa)
- Poor Transfer of Positively Charged Proteins
- Poor Semi-Dry Transfer
- Poor Transfer of a Wide Range of Protein Sizes
Band Smeared/Distorted
Possible Cause | Remedy |
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Membrane not uniformly wetted with methanol |
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Air bubbles under membrane and between other layers in the stack |
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Uneven contact between gel and membrane |
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Too much heat generated during the transfer |
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Filter paper dried out during semi-dry transfer |
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Proteins transferred too rapidly; protein buildup on the membrane surface |
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Possible Cause | Remedy |
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Autofluorescence from transfer membrane |
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Proteins passing through the membrane |
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Proteins retained in the gel |
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Isoelectric point of the protein is at or close to the pH of the transfer buffer |
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Poor detection when urea is used in the gel and/or transfer buffer |
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Incomplete transfer of proteins |
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Poor protein retention |
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Possible Cause | Remedy |
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No transfer of proteins |
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Possible Cause | Remedy |
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Insufficient protein retention SDS interferes with binding of small molecular weight proteins |
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Low methanol concentration in the transfer buffer |
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Insufficient protein binding time |
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Current doesn’t pass through the membrane |
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Possible Cause | Remedy |
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Methanol concentration is too high |
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Possible Cause | Remedy |
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Protein net charge in the transfer buffer is positive; proteins move toward cathode. |
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Possible Cause | Remedy |
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Current bypasses the gel stack |
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Possible Cause | Remedy |
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Different conditions required to transfer large and small proteins |
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