Transfer of Proteins

In this step the proteins are transferred from the gel to a blotting membrane driven by an electric field. Nitrocellulose and PVDF (Polyvinylidene fluoride) membranes are the most common membrane types for Western blotting.

Membrane types

Nitrocellulose membranes are made from nitrated cellulose and bind proteins mainly through hydrophobic interactions and hydrogen bonding.

Advantages

• Low background → often gives very clean blots
• Low autofluorescence (important for fluorescent read-outs)
• Good signal-to-noise ratio
• No pre-activation needed (unlike PVDF)
• Typically less expensive
• Easy to handle and cut

Disadvantages

• Lower protein binding capacity (~80–100 µg/cm²)
• More fragile — can tear or crack when dry
• Not ideal for repeated stripping and reprobing
• Slightly less durable for long-term storage

PVDF is a hydrophobic, synthetic polymer membrane with very high protein-binding capacity. It must be pre-wetted in methanol before use.

Advantages

• High protein binding capacity (~170–200 µg/cm²)
• More durable and mechanically strong
• Better for low-abundance proteins
• Works well for multiple stripping and reprobing cycles
• Superior for N-terminal sequencing or mass spectrometry applications

Disadvantages

• Requires methanol activation step
• Can produce higher background if blocking isn’t optimized
• Special low fluorescent variants have to be used for fluorescent detection systems
• More expensive than nitrocellulose
• More hydrophobic → sometimes higher nonspecific binding

Pore size

Both membrane types are available in two typical pore sizes.

• 0.45 µm → standard choice for most proteins
• 0.2 µm → better for small proteins (<20–25 kDa) to prevent loss through the membrane

Blotting systems

Semi-Dry Blotting: This is a very economic method (time and consumables) and widely used standard approach that can be used for a broad range of proteins. In a horizontal setup the gel–membrane sandwich is placed between buffer-soaked filter papers and sandwiched between plate electrodes. It requires less buffer and is faster than wet transfer. However, it may be less efficient for very large proteins and can be more sensitive to drying artifacts if not carefully controlled.

Wet (Tank)-Blotting: The gel and membrane are assembled in a transfer “sandwich” submerged in transfer buffer within a tank. An electric field drives proteins from the gel onto the membrane. This method is highly efficient and reliable, particularly for large proteins, but it is time-consuming and requires relatively large buffer volumes.

Important

In general transfer parameters are optimized for each blotting device. It is important to follow the manufacturer's instructions.

Check of transfer

The transfer efficiency can be evaluated by using reversible staining solutions like Ponceau red or commercial products compatible with the blotting membrane.