3.2.1 Membrane Transfer Methods

Common Membrane Transfer Methods

There are two common membrane transfer methods: semi-dry transfer and wet transfer.
Semi-dry transfer: It has a relatively short transfer time and achieves good results for proteins with small molecular weights. However, due to the small amount of buffer in the system, it is not suitable for proteins with large molecular weights.
Wet transfer: It has a relatively longer transfer time but enables complete protein transfer. Wet transfer is recommended for proteins with large molecular weights.
After membrane transfer, appropriate developing solutions can be used to detect the transfer effect. Common developing solutions include Ponceau S, Coomassie Brilliant Blue, Amido Black, etc.
Experimental Procedures
1. Semi-dry Transfer
Semi-dry transfer is faster than wet transfer and consumes less buffer.
The membrane is in direct contact with the gel, and several layers of buffer-wetted filter papers are placed above and below the gel and the membrane respectively.
A "sandwich" structure consisting of filter paper, gel, membrane, and filter paper is formed and placed in an electric field.
Generally, semi-dry transfer has lower efficiency than wet transfer, especially for proteins with large molecular weights.
When the electrode plates absorb heat, the heat generation has a relatively small impact on semi-dry transfer. However, prolonged semi-dry transfer time should be avoided, as this may lead to overheating and gel drying due to buffer depletion.
The filter papers and membrane should be cut to a size that is a few millimeters smaller than the length and width of the gel.
Ensure that the filter papers and membrane do not overlap with the gel; otherwise, it may cause system short-circuiting or result in inefficient or uneven protein transfer.
Semi-dry Transfer System
Semi-dry transfer is performed under constant current conditions (0.8 mA/cm²) and is relatively fast, taking approximately 15-45 minutes.
Note:
When conducting electrotransfer, ensure no air bubbles are formed when attaching the gel to the membrane. Air bubbles will cause blank spots on the membrane where no protein is transferred.
Pre-cooled transfer buffer can be used to prevent overheating of the system during transfer.
If you are unsure whether the "sandwich" assembly or power connection is correct, place a membrane on both sides of the gel in advance to avoid protein loss.
A colored marker pen can help you identify which membrane the protein is transferred to!
Small-molecular-weight proteins can penetrate the transfer membrane, so two membranes can be used simultaneously for transferring small-molecular-weight proteins.
2. Wet Transfer
In wet transfer experiments, both the gel and the membrane are completely immersed in the transfer buffer, and the direction of the current is from the gel to the membrane.
Usually, wet transfer requires a cooling unit, and a magnetic stir bar is used for internal circulation of the transfer buffer.
Wet transfer is recommended for proteins with large molecular weights, but it has a slower transfer speed and requires a large amount of buffer.
For clear and sharp bands and high-quality transfer results, wet transfer is the first choice, followed by semi-dry transfer.
During wet transfer, the large amount of buffer will heat up, and the temperature rise can cause irreversible denaturation of proteins. Therefore, it is very important to use cooled buffer and maintain a low temperature. Many commercial wet transfer systems can be connected to a constant-temperature circulating water bath.
Another common practice is to conduct the entire wet transfer experiment at 4°C, such as in a refrigerator.
Protein Molecular Weight and Transfer Conditions
Constant current: 200-350 mA for 70 minutes
Constant voltage: 70-120 V for 90 minutes
Tank (Wet) Transfer System
Note: The above membrane transfer conditions are for reference only; actual conditions should be adjusted according to the protein size.