Western blotting: Nitrocellulose Vs Polyvinylidene Difluoride (PVDF)

Western blotting is one of the most commonly performed experiments using antibodies and this week we’re going to be looking at a frequently asked question: should I use a nitrocellulose or Polyvinylidene Difluoride (PVDF) membrane?

We’ll be start from the beginning with a small overview of what Western blotting actually is before diving into what the literature has to say about the use of these membranes.

What is western blotting?

Western Blot Example ImageFirst described in 1979 by Towbin et al. [1,2], Western blotting is a commonly used technique used in the separation and identification of proteins. A sample mixture of proteins is first separated by it’s molecular weight through electrophoresis. The proteins are then transferred to a solid phase wherein they can then be probed with antibodies and their identity confirmed.

For a more in depth look at Western blotting, we found these resources particularly useful: Western blot: technique, theory, and trouble shooting. by Mahmood et al. or for a more visual explanation, the Journal of Visualised Experiments provides a number of instructional videos: Western Blotting: Sample Preparation to Detection[4] and Immunoblot Analysis[5].

What is the membrane used for?

Once your protein mixture has been separated, the resulting proteins need to be transferred to a membrane in order to make them accessible to antibodies for probing. This is normally achieved using a sandwich of the gel, membrane and electrodes and applying an electric field in order to draw the proteins into the membrane [3].

What are the advantages/disadvantages of each type of membrane?

Nitrocellulose
The good:
 – Has a high affinity for protein and therefore has high retention.
 – Is often cheaper than PVDF membranes [6]
 – Nitrocellulose is easily wetted avoiding the use of methanol.

The bad:
 – Unsupported nitrocellulose is innately fragile and is not recommended for stripping and reprobing [7], however, various versions of nitrocellulose are available which are designed for stripping and reprobing.
 – The use of methanol in transfer buffers (often as a way to remove SDS) has the effect of reducing the pore size of the gel, which can restrict the transfer of some molecules [6].

Polyvinylidene Difluoride (PVDF)
The good:
 – Membranes offer higher mechanical strength and allow for re-probing and storage.
 – Membranes have a higher binding capacity (150-160 µg/cm2) than nitrocellulose (80-100 µg/cm2) [7].
 – PVDF is hydrophobic and so lends itself well to the analysis of hydrophobic proteins.

The bad:
 – Background staining may be higher and so careful washing is required.
 – Requires pre-soaking with methanol
 – Chicken antibodies bind more readily to nylon-based membranes leading to high background signal [8].

After all that, having weighed up the pros and cons of each, and after speaking to a number of PhD students from various labs, what appears to play the biggest part in choosing a membrane is personal preference!
Preferences which are often influenced or based on what membrane people were first taught with or are available in the lab. Once established in a lab and protocols and buffers optimised for a particular membrane, switching would require re-optimisation, something to bear in mind if you’re thinking of swapping.

So there we have it, nitrocellulose versus PVDF membranes; How have you found different membranes to affect your blots? Do you have any tips or tricks in general to get the best out of your westerns? We’d love for you to join in the conversation either in the comments here or on Twitter, using our handle @CiteAb.

~ Matt and the CiteAb team

 


Image Credits

Image modified from FIGURE 7: Test cell lines
Frisa PS, Jacobberger JW Cell cycle-related cyclin b1 quantification. PLoS One. 2009 Sep 18;4(9):e7064. doi: 10.1371/journal.pone.0007064.


References

1 – Wu L, Hu X, Tang H, Han Z, Chen Y. Valid application of western blotting. Mol Biol Rep. 2014 May;41(5):3517-20. doi: 10.1007/s11033-014-3215-5. Epub 2014 Feb 8.
PMID : 24510387

2 – Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4.
PMID: 388439

3 – Mahmood T, Yang PC. Western blot: technique, theory, and trouble shooting. N Am J Med Sci. 2012 Sep;4(9):429-34. doi: 10.4103/1947-2714.100998.
PMID: 23050259

4 – Eslami, A., Lujan, J. Western Blotting: Sample Preparation to Detection. J. Vis. Exp. (44), e2359, doi:10.3791/2359 (2010).

5 – Gallagher, S., Chakavarti, D. Immunoblot Analysis. J. Vis. Exp. (16), e759, doi:10.3791/759 (2008).

6 – Factors Influencing Western Blot Results. Accessed: 2nd July 2014

7 – Membranes and Blotting Papers. Accessed: 2nd July 2014

8 – WESTERN BLOTTING – A BEGINNER’S GUIDE. Accessed: 2nd July 2014


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