Protein Transfer from Gel to Membrane in Western Blot Assay

Following gel electrophoresis, the separated protein mixtures are transferred to a solid support for further analysis. Due to the speed and efficiency of transfer, electroblotting is the method relied upon in most laboratories. Transfer is performed by passing a current across the gel to the membrane. There are two common membrane types used for western blot (WB) analysis, polyvinylidene difluoride (PVDF) and nitrocellulose (NC). Compared to NC membrane, PVDF membranes offer better protein retention, physical strength, and chemical compatibility. In general, PVDF is much better for low molecular weight (MW) proteins.

Principle

The gel and membrane are assembled into a sandwich along with several sheets of filter paper which protect the gel and membrane and help to ensure close contact between their surfaces. Taking PVDF membrane for example, the PVDF membrane is placed between the gel and the positive electrode so that the negatively charged proteins migrate from the gel onto the PVDF membrane. The transfer buffer used for electroblotting is similar to gel running buffer with the addition of methanol which helps proteins bind to the blot. Electrophoretic transfer can be accomplished under wet or semi-dry conditions. In a wet transfer, the gel/blotting paper/filter paper sandwich is placed into a cassette along with protective fiber pads.

Size of The Target Protein

The size of the target protein should be considered when choosing transfer conditions. Smaller proteins will transfer out of the gel faster and may actually transfer through the PVDF membrane into the filter papers beyond. PVDF with a smaller pore size can be used for small proteins and peptides. If there is a suspicion that the protein is transferring through the PVDF membrane, then a second membrane can be included behind the first to catch proteins that migrate through. In contrast, large proteins can be slower to elute and may be retained within the gel, so overnight wet transfer is usually preferred.

Common Transfer Buffer Recipes

1. 1X Transfer Buffer (Wet)
• 25 mM Tris base
• 192 mM glycine
• 20 % methanol
• Adjust pH to 8.3
2. 1X Transfer Buffer (Semi-Dry)
• 48 mM Tris base
• 39 mM glycine
• 20 % methanol
• Adjust pH to 8.3

Protein Transfer Protocol

1. Prepare the PVDF membrane: 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.
2. Soak filter papers and sponges in the transfer buffer for 5-10 mins prior to assembly of the transfer sandwich.
3. Once electrophoresis is complete, remove the gel from the electrophoresis apparatus and equilibrate it by soaking it in transfer buffer for 5-10 mins.
4. Prepare the sandwich according to the illustration below. Sequentially assemble the layers of the sandwich. Gently remove any air bubbles with pipette.

Note: Bubbles between the gel and the membrane will inhibit the transfer of proteins to the membrane.

5. Place the sandwich into a transfer cassette and perform semi-dry/wet transfer. For wet transfer, the gel side of the cassette holder should face the cathode (-) while the membrane side should face the anode (+). For semi-dry transfer, the gel side should face the cathode plate (-), while the membrane side should face the anode plate (+). Set the parameters and time.

Important Tips

1. Avoid touching the membrane with your fingers. Proteins on fingers will block efficient transfer and create dirty blots.
2. After sandwiching the gel and membrane between paper, air bubbles between the gel and membrane can be removed by rolling them out with a pipette, roller, a 15 mL tube, or by assembling the sandwich in a dish of transfer buffer to prevent formation of bubbles in the first place.
3. Make sure the paper and membrane are cut to the same size as the gel. Large overhangs may prevent a current from passing through the membrane in semi-dry transfers.
4. Chicken antibodies tend to bind PVDF, leading to high background. Switching to NC membrane.

Do you have any suggestions on how to perform a successful WB experiments? Do you have questions on a particular step in the protocol? Find what you need from the quick links below.

More WB Resource

Western Blot Protocols & Troubleshooting & Guide

Western Blot Protocol

Western Blot Troubleshooting

Sample Preparation in Western Blot Assay

Running an SDS-PAGE Gel in Western Blot Assay

Western Blot Illustrated Assay

Stripping and Reprobing Protocol

Western Blot Storage Protocol