Related Resources: Brochures | Application NotesElectrotransfer 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:
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
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
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.
Poor Transfer of Large Molecular Weight Proteins (~ >80 kDa)
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.