Protein transfer from gel to membrane in western blot



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Sample preparation

Running the SDS-PAGE gel

Transferring protein from gel to membrane

Immunoblotting and detection

Membrane stripping and reprobing

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PROTEIN TRANSFER

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 is generally better for low molecular weight proteins. This membrane can be purchased in different pore sizes. For proteins less than 30 kD, the pore size of 0.2 µM PVDF 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

  1. Prepare the 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.

  2. Note: Do not wet nitrocellulose membranes with methanol or the membrane will dissolve.


    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


    Tip: Adding up to 0.05% SDS in the transfer buffer can improve transfer efficiency in some cases.


  3. Soak filter papers and sponges in the transfer buffer for 10 mins prior to assembly of the transfer sandwich.

  4. Once electrophoresis is complete, remove the gel from the electrophoresis apparatus and equilibrate it by soaking it in transfer buffer for 10 mins.

  5. Prepare the 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.


  6. Place the sandwich into a transfer cassette and perform semi-dry or wet (also known as tank) transfer according to the manufacturer’s instructions of the blotting apparatus. 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 (+).

  7. Semi-dry transfer: generally faster, better suited for larger proteins greater than 100 kDa. Commonly used transfer time: 60 mins at 25V.


    Wet transfer: recommended for smaller proteins, especially proteins smaller than 30 kDa. Commonly used transfer time: 1 hr at 100V at 4 ˚C


    TIP: 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.



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