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
Membrane not uniformly wetted with methanol	The entire membrane must be pre-wet with methanol; the entire membrane should change uniformly from opaque to semi-transparent.
Air bubbles under membrane and between other layers in the stack Effect of air bubbles between gel and membrane during Western blot transfer step	Using a pipette or stirring rod, gently roll out any trapped air bubbles while assembling the stack.
Uneven contact between gel and membrane	Make sure entire gel and membrane surfaces are in good contact.
Too much heat generated during the transfer	The temperature of the run should not exceed 20 °C. For a tank transfer, pre-chill the buffer or carry out the transfer in a cold room. For a semi-dry transfer, either shorten the run time, increase the number of filter papers, or reduce the current.
Filter paper dried out during semi-dry transfer	Make sure filter paper is thoroughly drenched prior to transfer or use additional sheets. Be sure the stack is assembled in less than 15 minutes.
Proteins transferred too rapidly; protein buildup on the membrane surface	Reduce the strength of the electrical field.

High Background in Fluorescent Detection

Possible Cause	Remedy
Autofluorescence from transfer membrane	Use low-autofluorescence membrane, such as Immobilon®-FL membrane, for transfer. Reverse image demonstrating fluorescent detection of transferrin in human serum on various Western blot transfer
Proteins passing through the membrane	Increase the time the proteins have to interact with membrane by reducing the voltage by as much as 50%. Highly negatively charged proteins (due to high aspartic acid and glutamic acid content) tend to move very fast in an electric field. Decrease the voltage to slow down migration of these proteins. Presence of SDS in the gel may inhibit protein binding. Equilibrate the gel in the transfer buffer for at least 15 minutes Gel (top) and blot (bottom) show that equilibration improves transfer efficiency for Western blot. Methanol concentration in transfer buffer is too low to facilitate removal of SDS. Increase the methanol to 15 – 20%, especially for smaller molecular weight proteins. The membrane must be pre-wet with methanol; the entire membrane should change uniformly from opaque to semi- transparent. Switch to Immobilon®-PSQ transfer membrane.
Proteins retained in the gel	If the methanol concentration in the transfer buffer is too high, it can remove SDS from proteins and lead to protein precipitation in the gel. This would reduce the transfer of large molecular weight proteins out of the gel. If protein precipitation is an issue, the transfer buffer can be supplemented with SDS (0.01% – 0.05%) to aid in solubility. In addition, excess methanol can tend to shrink or tighten a gel, thus inhibiting transfer of large molecular weight proteins.
Isoelectric point of the protein is at or close to the pH of the transfer buffer	A protein that has the same isoelectric point as the pH of of the transfer buffer will have no net charge and thus will not migrate in an electric field. To facilitate transfer, try a higher pH buffer such as 10 mM CAPS buffer at pH 11, including 10% methanol or a lower pH buffer such as an acetic acid buffer.
Poor detection when urea is used in the gel and/or transfer buffer	Reduce the temperature by using a circulating buffer setup or run your transfer in a cold room. Urea in the presence of heat can cause carbamylation of proteins, which can change the charge of amino acids in a protein. This could affect the epitopes essential for antibody recognition and binding.
Incomplete transfer of proteins	Stain the gel to check for residual proteins. If transfer was not complete, review your transfer technique.
Poor protein retention	Once transfer is complete, be sure to dry the membrane completely to obtain optimal binding and fixation of the proteins. This should be done prior to any downstream detection method.

No Signal

Possible Cause	Remedy
No transfer of proteins	Check for the gel and membrane orientation during the transfer process. Use pre-stained molecular weight standards to monitor transfer.

Poor Transfer of Small Molecular Weight Proteins

Possible Cause	Remedy
Insufficient protein retention SDS interferes with binding of small molecular weight proteins	Switch to Immobilon®-PSQ transfer membrane. Remove SDS from the transfer solution.
Low methanol concentration in the transfer buffer	Use higher percentage of methanol (15% – 20%) in the transfer buffer.
Insufficient protein binding time	A lower voltage may optimize binding of small proteins to the membrane.
Current doesn’t pass through the membrane	Cut membrane and blotting paper exactly to the gel size; do not allow overhangs. Explore pre-cut membrane and filter paper sandwiches

Poor Transfer of Large Molecular Weight Proteins (~ >80 kDa)

Possible Cause	Remedy
Methanol concentration is too high	Reducing the methanol concentration to 10% (v/v) or less should help in the transfer of large molecular weight proteins by allowing the gel to swell. Moreover, a lower methanol percentage would also reduce SDS loss from the proteins and reduce protein precipitation in the gel. Proteins >200 kDa are not as sensitive to interference from the SDS in binding to membrane as are proteins <100 kDa.

Poor Transfer of Positively Charged Proteins

Possible Cause	Remedy
Protein net charge in the transfer buffer is positive; proteins move toward cathode.	Reverse the transfer stack such that the Immobilon® transfer membrane is on the cathode side of the gel.

Poor Semi-Dry Transfer

Possible Cause	Remedy
Current bypasses the gel stack	Make sure the membrane and blotting paper are cut exactly to the gel size and there are no overhangs. Explore pre-cut membrane and filter paper sandwiches

Poor Transfer of a Wide Range of Protein Sizes

Possible Cause	Remedy
Different conditions required to transfer large and small proteins	Refer to “Transfer of a broad MW range of proteins may require a multi-step transfer” (T. Otter et al., Anal. Biochem. 162:370-377 (1987). Use three-buffer system for semi-dry transfer (View protocol on page 36 of the Protein Blotting Handbook (PDF).)