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Insane in the Membrane! PVDF vs. Nitrocellulose – Which One Comes Out on Top?

When it comes to Western blotting, there’s no denying it: Your membrane is a key player. After all it is the physical scaffold that holds your precious samples and it needs to be up to the challenges you throw at it. But depending on your protein’s properties and your downstream detection steps, finding the optimal membrane may take a bit of trial and error. To help you choose, I will pit two membrane heavyweights against each other, PVDF vs. Nitrocellulose. Find out which one comes out on top:

 NitrocellulosePVDF
What is it?Nitrocellulose (either alone or attached to an inert scaffold)Polyvinylidine difluoride
How protein-loving is it?
(i.e. what is the protein binding capacity, μg/cm2)
80–100170–200
What do people use it for?Western blotting (especially low molecular weight proteins <20kDa)

Nucleic acid analysis (fragments <300bp)

Amino acid analysis

Dot/slot blotting
Western blotting (especially high molecular weight proteins)

Protein sequencing

Amino acid analysis

Solid phase assay systems
How do proteins stick?Binding interactions are likely hydrophobicProteins bind by hydrophobic and dipole interactions
Physical characteristicsBrittle and fragilePhysically durable
Does it come in different pore sizes?Yes. Both nitrocellulose and PVDF come in typical pore sizes of 0.1, 0.2 or 0.45μm. The 0.45μm membrane is suitable for most protein blotting applications but small peptides or low molecular weight proteins may transfer better onto 0.1 or 0.2μm pore size membrane
Chemically resistant?NoYes – suitable for sequencing applications
How sensitive is it?Nitrocellulose membranes have high protein-binding affinity but are not capable of the detection sensitivity of PVDF membranes. On the plus side, you may get lower background signal if using nitrocellulosePVDF’s higher binding capacity allows for greater sensitivity in detection of lowly expressed proteins, but it also means that you can get higher background when it comes to your antibody detection steps
Can it be stripped and reprobed?Proceed with caution. It is difficult to strip and reprobe nitrocellulose membranes without loss of signalYes. There is better retention of adsorbed proteins due to PVDF’s greater hydrophobicity, allowing membranes to be stripped and reprobed easily. Check out this article for more tips on how to strip and reprobe
Is methanol required?Yes. Methanol must be used in the transfer buffer for nitrocellulose membranes – this can cause precipitation of high molecular weight proteinsNo. The membrane must be pre-wetted with methanol before use but can then be used with transfer buffers that contain no methanol. It can be a useful technique for high molecular weight proteins that precipitate in low SDS/high methanol solutions.
Expert tip: be sure to equilibrate your membrane before transfer if you choose to do this!
What about SDS?Doesn’t require SDS in transfer buffer. This enhances the binding of low molecular weight proteins and helps prevent protein “blowout”, i.e. over-transfer through the membraneBlotting efficiency onto PVDF is significantly enhanced in the presence of SDS

So which membrane emerges victorious for your experiments?

3 Comments

  1. Allie Burns on June 25, 2015 at 3:55 pm

    The PVDF column looks like it should have a link to an article on stripping and re-probing membranes but I couldn’t find a link. Where is that article? I’m interested in reading it. Thanks!

    • Amanda Welch on June 26, 2015 at 2:12 am

      Great catch! The column has been updated to include the link to the article on stripping and reporting membranes.

      • Anna on April 28, 2016 at 9:04 pm

        Is there any published data on which information in this article is based? Would you have links for this literature?
        Thank you

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