Commonly used transfer time: 1 hour at 100V at 4☌. Wet transfer: recommended for smaller proteins, especially proteins Commonly used transfer time: 1 hour at a constant current (1.25 mA/cm2). Semi-dry transfer: generally faster, better suited for larger proteins >100 kDa.Place the sandwich into a transfer cassette and perform semi-dry or wet transfer according to the manufacturer’s instructions of the blotting apparatus.Bubbles between the gel and the membrane will inhibit the transfer of proteins to the membrane.

Gently remove any air bubbles with a roller or pipette. Sequentially assemble the layers of the sandwich. Prepare the transfer sandwich according to the illustration below.After electrophoresis, remove the gel from the electrophoresis apparatus and equilibrate it by soaking in 1X Transfer Buffer for 10 minutes.Soak filter papers and sponges in 1X Transfer Buffer for 10 minutes prior to assembly of the transfer “sandwich”.Note: PVDF membrane has a higher binding capacity, best for low expressed proteins, but nitrocellulose membrane is ideal for lower molecular weight proteins. DO NOT wet nitrocellulose membranes with methanol or the membrane will dissolve. Note: PVDF membrane must be wet in methanol but can use methanol-free transfer buffer. Handle the membrane carefully, ideally with rounded tweezers to avoid scratching or puncturing the surface. Prepare PVDF membrane by wetting it in methanol for 30 seconds and then soaking it briefly in distilled water followed by 1X transfer buffer.

Additionally, PVDF membranes offer better protein retention, physical strength, and chemical compatibility compared to nitrocellulose. For proteins less than 30 kD, the pore size of 0.2 µM is recommended over the 0.45 µM pore size. PVDF is best for higher molecular weight proteins whereas nitrocellulose is best for low-to-mid molecular weight proteins. Both membranes can be purchased in different pore sizes. PVDF has a higher binding capacity and is generally better for lowly expressed proteins. There are two common membrane types used for western blot analysis: PVDF and nitrocellulose. Transfer is performed by passing a current across the gel to the membrane. Transferring protein from gel to membraneĪfter electrophoresis is complete, proteins must be transferred from the gel onto a suitable membrane for antibody staining and detection.

Stripping membrane pro con