3.4 Experimental Tips

Common Membrane Transfer Methods
There are two commonly used membrane transfer methods: semi-dry transfer and wet transfer.
Semi-dry transfer has a relatively short transfer time and works well for small-molecular-weight proteins. However, due to the small amount of buffer in the system, it is not suitable for large-molecular-weight proteins.
Wet transfer requires a relatively longer transfer time but enables complete protein transfer. Wet transfer is recommended for large-molecular-weight proteins.
After membrane transfer, an appropriate staining solution can be used to verify the transfer efficiency. Commonly used staining solutions include Ponceau S, Coomassie Brilliant Blue, and Amido Black.

Laboratory Tips
1. For Large-Molecular-Weight Proteins
Add 0.1% SDS to the transfer buffer.
Reduce the methanol concentration to 10% or even lower.
Use 4°C overnight wet transfer instead of semi-dry transfer.
The transfer time for large-molecular-weight proteins can be appropriately extended to 2–3 hours, and the current can be adjusted to approximately 100 mA.
2. For Small-Molecular-Weight Proteins
Completely remove SDS from the buffer.
Maintain the methanol concentration at 20%.
Use a membrane with a small pore size.
The transfer time for small-molecular-weight proteins can be shortened to 30 minutes.
3. Determining the Membrane Orientation
After transfer, cut a corner of the membrane as a marker to distinguish the front and back sides; alternatively, use a pencil to make marks or ensure the protein Marker is arranged asymmetrically during electrophoresis for orientation identification.
4. High Background After Membrane Transfer
Select NC (nitrocellulose) membrane.
5. Contamination During Membrane Transfer
Avoid touching the membrane with fingers; use forceps to handle the membrane. Oils and proteins on fingers can contaminate the membrane.
6. Size of Filter Paper
Ensure the size of the filter paper and membrane matches that of the gel. When using semi-dry transfer, excessive filter paper/membrane edges will impede the current from passing through the membrane.