Key Difference – Nitrocellulose vs PVDF
Western blotting is a method which allows detection and quantification of specific proteins from a protein sample. Reliability of the technique depends on the selection of a correct membrane for absorption of proteins from the gel. There are different types of microporous membranes. Nitrocellulose and PVDF membranes are two such membranes preferred by the researchers due to their special attributes over the other types of membranes. The selection between the nitrocellulose or PVDF is also another challenge in western blotting. Both nitrocellulose and PVDF have a high protein absorption capacity. The key difference between nitrocellulose and PVDF membrane is that nitrocellulose membranes do not have the ability to strip off antibodies and reusing the membrane for antibody reprobing while PVDF membranes have stripping and reusing ability.
CONTENTS
1. Overview and Key Difference
2. What is Nitrocellulose
3. What is PVDF
4. Side by Side Comparison – Nitrocellulose vs PVDF
5. Summary
What is Nitrocellulose?
Nitrocellulose is a polymer manufactured by treating cellulose with nitric acid and is used to make microporous membranes in molecular biology, especially for blotting techniques such as southern, northern and western blotting. Pore sizes of nitrocellulose membranes range from 3 to 20 µm. Nitrocellulose microporous membranes facilitate the detection of immunochemical reaction happening on the surface of the membrane. Therefore, nitrocellulose membranes are frequently used for immobilization of proteins and detection of specific proteins in western blotting. Nitrocellulose membranes can also immobilize glycoproteins and nucleic acids.
Nitrocellulose membranes are preferred in lateral flow assays due to several features. Nitrocellulose membranes absorb proteins at a high concentration. The solvent used to wet the membrane does not diminish the nitrocellulose membrane protein absorption. Nitrocellulose membranes can be easily cut to the desired gel size and transfer proteins from the gel to the membrane by electric or capillary transfer. Nitrocellulose allows faster flow of proteins through the membrane with high binding potential. Nitrocellulose shows an improved strength of handling. Another special attribute of nitrocellulose membrane is that it can be easily glued with non-solvent water resistant adhesives onto various plastic backings.
Figure 01: Nitrocellulose membrane for western blotting
What is PVDF?
Polyvinylidene difluoride (PVDF) is fluoropolymer produced by the polymerization of vinylidene difluoride and has a high immobilization capability of proteins. Therefore, microporous membranes made from PVDF are used in western blotting techniques to analyze specific proteins from the mixtures of proteins. PVDF membranes can also be used for amino acid analysis and protein sequencing. The most important characteristic of PVDF membrane over nitrocellulose membrane is that it can be easily stripped off the antibodies and reused for subsequent antibody probes.
PVDF membranes are thicker than nitrocellulose membranes; hence, more resistant to damages during reuse. PVDF membranes are highly hydrophobic. Therefore, they must be soaked in methanol or isopropanol before use.
What is the difference between Nitrocellulose and PVDF?
Nitrocellulose vs PVDF |
|
| Nitrocellulose is a polymer composed of cellulose. | PVDF is a fluoropolymer produced by the polymerization of vinylidene difluoride. |
| Membrane Pore Size | |
| Typical pore sizes are 0.1, 0.2 or 0.45μ | Typical pore sizes are 0.1, 0.2 or 0.45μm |
| Protein Binding Capacity | |
| Nitrocellulose has a protein binding capacity of 80 to 100 μg/cm2. | PVDF has a protein binding capacity of 170 to 200 μg/cm2. |
| Sensitivity | |
| This has a low sensitivity compared to PVDF. | This has a high sensitivity. |
| Detection of Lowly Expressed Proteins | |
| Since the sensitivity is low in nitrocellulose membranes, it is not suitable for detection of lowly expressed proteins. | This is more suitable for the detection of lowly expressed proteins due to its high sensitivity. |
| Background Noise | |
| This has lower background noise | This has higher background noise. |
| Interactions with Proteins | |
| Protein molecules bind to nitrocellulose membranes through hydrophobic interactions. | Proteins bind to PVDF membranes through hydrophobic and dipole interactions. |
| Nature of the Membrane | |
| Nitrocellulose is brittle and fragile. However, versions of nitrocellulose are available, and they are resistant. | PVDF is more durable and has higher chemical resistance. |
| Ability to Strip and Reuse | |
| Nitrocellulose may have difficulty in stripping and reprobing without losing the signal. | PVDF is ideal for reprobing and sequencing applications. |
| Suitability | |
| Nitrocellulose is ideal for detecting low molecular weight proteins. | PVDF is more suitable for detecting higher molecular weight proteins. |
| Other Uses | |
| Nitrocellulose can be used for nucleic acid analysis and dot/slot blotting. | PVDF can be used for protein sequencing and solid phase assay systems. |
| Cost | |
| This is cheaper than PVDF membranes. | This is more expensive than nitrocellulose membranes. |
| Need for Pre-wetting | |
| Nitrocellulose membranes do not require presoaking with methanol | PVDF membranes require presoaking with methanol. |
Summary – Nitrocellulose vs PVDF
Nitrocellulose membranes were the first membranes used commercially for lateral flow assay. They have a high capacity for absorbing proteins. Therefore, nitrocellulose membranes are used in western blotting. PVDF is another type of membrane used in western blotting and it also has a high protein absorption capacity. Both types are used in western blotting for protein analysis. However, PVDF membranes have more special attributes, making them more suitable than nitrocellulose membranes for western blotting. But, nitrocellulose membranes are more suitable for detecting low molecular weighted proteins, PVDF membranes are more suitable for detecting high molecular weighted proteins. This is the difference between nitrocellulose and PVDF membranes.
Image Courtesy:
1.”Western blot transfer” By Bensaccount at English Wikipedia (CC BY 3.0) via Commons Wikimedia
Reference:
1.Luque-Garcia, Jose L., Ge Zhou, Tung-Tien Sun, and Thomas A. Neubert. “Use of Nitrocellulose Membranes for Protein Characterization by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.” Analytical chemistry. U.S. National Library of Medicine, 15 July 2006. Web. 28 Mar. 2017
2.”Molecular Markers and Plant Biotechnology.” Google Books. N.p., n.d. Web. 28 Mar. 2017
3.Monday, April 8, 2013 Tweet. “The Magic’s In the Membrane: Choose Wisely for Best Western Blot Results.” Biocompare. N.p., 08 Apr. 2013. Web. 28 Mar. 2017