After electrophoresis is complete, proteins must be transferred from the gel onto a suitable membrane for antibody staining and detection. Transfer is performed by passing a current across the gel to the membrane. There are two common membrane types used for western blot analysis: PVDF and nitrocellulose. PVDF has a higher binding capacity and is generally better for lowly expressed proteins. Both membranes can be purchased in different pore sizes. PVDF is best for higher molecular weight proteins whereas nitrocellulose is best for low-to-mid molecular weight proteins. For proteins less than 30 kD, the pore size of 0.2 µM is recommended over the 0.45 µM pore size. Additionally, PVDF membranes offer better protein retention, physical strength, and chemical compatibility compared to nitrocellulose.
Protein transfer protocol
Prepare PVDF membrane by wetting it in methanol for 30 seconds and then soaking it briefly in distilled water followed by 1X transfer buffer. Handle the membrane carefully, ideally with rounded tweezers to avoid scratching or puncturing the surface.
Note: PVDF membrane must be wet in methanol but can use methanol-free transfer buffer. DO NOT wet nitrocellulose membranes with methanol or the membrane will dissolve.
Note: PVDF membrane has a higher binding capacity, best for low expressed proteins, but nitrocellulose membrane is ideal for lower molecular weight proteins.
Buffer
Components
1X Transfer Buffer (wet)
25 mM Tris base
192 mM glycine
20% methanol
Adjust pH to 8.3
1X Transfer Buffer (semi-dry)
48 mM Tris base
39 mM glycine
20% methanol
Adjust pH to 8.3
Soak filter papers and sponges in 1X Transfer Buffer for 10 minutes prior to assembly of the transfer “sandwich”.
After electrophoresis, remove the gel from the electrophoresis apparatus and equilibrate it by soaking in 1X Transfer Buffer for 10 minutes.
Prepare the transfer sandwich according to the illustration below. Sequentially assemble the layers of the sandwich. Gently remove any air bubbles with a roller or pipette. Bubbles between the gel and the membrane will inhibit the transfer of proteins to the membrane.
Place the sandwich into a transfer cassette and perform semi-dry or wet transfer according to the manufacturer’s instructions of the blotting apparatus.
Semi-dry transfer: generally faster, better suited for larger proteins >100 kDa. Commonly used transfer time: 1 hour at a constant current (1.25 mA/cm2).
Wet transfer: recommended for smaller proteins, especially proteins <30 kDa. Commonly used transfer time: 1 hour at 100V at 4°C.
Transfer time/voltage may require optimization. Over-transferring (or pulling protein all the way through the membrane) can occur and thus caution must be taken, especially for small proteins.
