Transfer Basics
The Importance Uses of Transfer Membrane
Protein Transfer on to a Membrane
Protein transfer is a vital step in western blot analysis which involves the transfer of proteins separated in a gel by electrophoresis to a solid support matrix. Immobilizing the protein to a solid support matrix facilitates the detection of specific proteins using antibodies directed against the protein(s) of interest. Typical solid matrices are membrane sheets of nitrocellulose and PVDF.
Blotting
After separating the protein mixture, it is transferred to a membrane. The transfer is done using an electric field oriented perpendicular to the surface of the gel, causing proteins to move out of the gel and onto the membrane. The membrane is placed between the gel surface and the positive electrode in a sandwich
Why transfer process is important?
Protein transfer from the gel to the membrane is necessary for two reasons:
- Better handling capability offered by the membrane compared to a fragile gel
- Better target protein accessibility on the membrane by macromolecules like antibodies
After transfer, the membrane must be blocked to prevent non-specific binding of the antibody to the membrane surface. The transferred protein is then probed sequentially with antibodies and detection probe (e.g., enzyme, fluorophore, isotope). An appropriate method is then used to detect the localized probe to document the location and relative abundance of the target protein.
The type of Membranes
There are two common membrane types used for western blot analysis: PVDF and nitrocellulose. These membranes are commonly used because they offer:
- Large surface area-to-volume area ratio
- High binding capacity
- Extended storage of immobilized macromolecules
- Ease of use
- The potential to be optimized for low background signal and reproducibility
