The main difference between nitrocellulose and PVDF membrane is that nitrocellulose membrane has a higher protein-binding capacity whereas PVDF membrane has a comparatively low protein-binding capacity. Furthermore, protein molecules bind to the nitrocellulose membrane through hydrophobic interactions while protein molecules bind to the PVDF membrane through both hydrophobic interactions and dipole interactions. In addition, the nitrocellulose membrane has a higher sensitivity than the PVDF membrane.
Nitrocellulose and PVDF membrane are two types of membranes used in western blot to transfer separated proteins by gel electrophoresis. Generally, the main function of a membrane in blotting is to serve as a physical scaffold, holding the protein fragments.
Key Areas Covered
1. What is Nitrocellulose Membrane
– Definition, Characteristics, Importance
2. What is PVDF Membrane
– Definition, Characteristics, Importance
3. What are the Similarities Between Nitrocellulose and PVDF Membrane
– Outline of Common Features
4. What is the Difference Between Nitrocellulose and PVDF Membrane
– Comparison of Key Differences
Nitrocellulose Membrane, Protein Binding Capacity, PVDF Membrane, Sensitivity, Western Blot
What is Nitrocellulose Membrane
Nitrocellulose membrane is one of the first types of membranes used for western blotting. It is still the most popular choice for this technique as it is instantaneous and nearly irreversible. Also, the nitrocellulose membrane is easily hydrated. However, it is not suitable for stripping, reprobing or harsh chemical treatments. The supported nitrocellulose membranes are one of the set choices as their inert support structure increases the strength and resilience.
Furthermore, in western blotting, protein fragments on the gel are transferred to the nitrocellulose membrane in a process called electrophoretic transfer. The principle behind this process is the migration of the negatively-charged proteins towards the anode.
What is PVDF Membrane
PVDF membrane is the other type of membrane used in western blotting. It has a higher protein-binding capacity when compared to the nitrocellulose membrane. Thus, this increases the sensitivity of the detection as well. However, the increased sensitivity results in a higher background due to the detection of lowly expressed proteins.
Moreover, concerning the nitrocellulose membrane, the PVDF membrane does not have to be pre-wet with methanol before use along with the transfer buffer. This prevents the precipitation of high molecular weight proteins by low SDS/high methanol solutions. Meanwhile, the presence of SDS in the transfer buffer increases the blotting efficiency in PVDF membranes.
Similarities Between Nitrocellulose and PVDF Membrane
- Nitrocellulose and PVDF membrane are two types of membranes used in western blot.
- They are important for the binding of separated protein fragments on the gel.
- Moreover, the transformation process occurs in a process called electrophoretic transfer.
- Proteins bind to the membrane mainly through hydrophobic interactions.
- Their typical pore sizes are 0.1, 0.2 or 0.45 μm. Basically, 0.1 and 0.2 μm pore sizes are important for small peptides and low molecular weight proteins less than 15 kDa. The 0.45 μm membrane is suitable for most protein blottings. On the other hand, for the quantification of proteins and when loaded in low concentrations, small pore sizes are better to use.
- Furthermore, pre-cut and pre-wetted membranes in methanol are ideal for convenience, reproducibility and high throughput applications.
- Both have significantly high protein binding capacity and sensitivity.
- They are also capable of resisting background and non-specific binding.
- Moreover, specific proteins can be detected by chemiluminescence and fluorescence methods.
Difference Between Nitrocellulose and PVDF Membrane
Nitrocellulose membrane refers to a sticky membrane used for the immobilization of proteins in western blot. Meanwhile, PVDF membrane refers to the other type of membrane used in western blot for protein transfer with high binding capacity.
The nitrocellulose membrane is brittle and fragile, while the PVDF membrane is physically durable.
While the nitrocellulose membrane is not chemically resistant, the PVDF membrane is chemically resistant.
The nitrocellulose membrane has to be pre-wetted with methanol while the PVDF membrane does not need to pre-wet.
Protein Binding Capacity
Moreover, the nitrocellulose membrane has a comparatively low protein binding capacity (80-100 μg/cm2) while the PVDF membrane has a high protein binding capacity (170-200 μg/cm2).
Interactions of Proteins
Proteins bind to the nitrocellulose membrane through hydrophobic interactions while they bind to the PVDF membrane through both hydrophobic and dipole interactions.
Nitrocellulose membrane does not require SDS in the transfer buffer, which enhances the binding of low molecular weight proteins and helps prevent the over transfer of proteins through the membrane while SDS enhances the blotting efficiency of proteins onto PVDF membrane.
Usage in Western Blotting
While the nitrocellulose membrane is used for the low molecular weight proteins, PVDF membrane is used for the high molecular weight proteins.
Furthermore, the nitrocellulose membrane has a comparatively low sensitivity while the PVDF membrane has high sensitivity due to its higher binding capacity.
Nitrocellulose membrane produces a lower background due to its less sensitivity while the PVDF membrane has a higher background due to its greater sensitivity in the detection of proteins with lower expression.
Stripping and Reprobing
In addition to these, the nitrocellulose membrane is difficult to strip and reprobe while the PVDF membrane is easy to strip and reprobe due to its higher hydrophobicity.
Nitrocellulose membrane is used for the analysis of nucleic acids (<300 bp), amino acid analysis, and dot/slot blotting while the PVDF membrane is used for protein sequencing, amino acid analysis, and solid-phase assay systems.
Nitrocellulose membrane is the type of membrane important in western blot. However, it has a comparatively low protein-binding capacity and sensitivity. It also produces a lower background. Moreover, it is mainly used in the separation of high molecular weight proteins. The PVDF membrane is the other type of membrane used in western blot for the separation of high molecular weight proteins. Moreover, this membrane has a comparatively higher protein-binding capacity and sensitivity. However, it produces a higher background. Therefore, the main difference between nitrocellulose and PVDF membrane is their protein-binding capacity and sensitivity.
1. The ProteinMan. “PVDF or Nitrocellulose – Which Membrane Is Best?” G-BIOSCIENCES, 12 Nov. 2014, Available Here.