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Drying Methods Protocol for PVDF

When binding nucleic acids or proteins, a high-binding material called PVDF (polyvinyl difluoride) membrane provides support for the antibodies. Through Western blotting, the membranes are stacked together, prepared for the antibody and then dried. Protocol for drying the membrane requires knowledge of how membranes work, cathode plates, anode plates and application of methanol solutions. With the correct protocol, the membrane safely binds with the specific antibody and is ready for immediate use or storage.

Preparing the Membrane
- Preparing the PVDF membrane cleans and stabilizes the membrane before the transfer. Before drying, the membrane is "blocked" to prevent binding from anything other than the specific nucleic acids, such as nonspecific antibodies. During this procedure, you wet the membrane by laying it on a flat surface covered in methanol. The membrane changes color during this phrase, from opaque to semitransparent. Once finished with the methanol bath, the membrane is placed in deionized water to soak for approximately two minutes. The membrane must then soak again in a methanol solution that is highly acidic.
Assembly of Membranes Before Transfer
- Two of the membrane papers are soaked in the first methanol solution and placed on an anode plate. During this process, you must carefully check for trapped air bubbles, which could compromise the transfer. Another piece of filter paper is soaked in the second methanol solution and placed on top of the stack. A gel then goes on top of the membrane, with care not to trap air bubbles between the gel application and membrane. Three new pieces of paper are then soaked in a cathode buffer and placed on top of the gel. If any air bubbling occurs, a test tube works to smooth out the membranes.
Transfer
- In this part of the process, you connect high-voltage cords to a power supply and apply a constant current to the gel area on the membrane for 30 minutes. Transfer times will vary depending on the type of antibody. After 30 minutes, you remove the power supply and transfer membrane. The lower right corner of the membrane is then cut to check orientation. The first two layers of the filter paper are discarded, including the gel. The bands of the molecular weight are marked with a ballpoint pen.
Drying
- Once the transfer is complete, you dry the membrane in one of two ways. For faster drying, the membrane is soaked in 100 percent methanol for 10 seconds to remove any water. The membrane then rests on a piece of paper towel for 15 minutes. For a slower drying method, the membrane is placed on the paper towel without adding methanol. Once dried, the membrane may be used or stored in a dry container at 4 degrees Celsius for future use.

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