Western blot membrane
PVDF vs Nitrocellulose (Western Blot Membranes)
If you don’t read this article you will miss out on the following:
- What type of membranes are used in a western blot
- Which western blot membrane is better and why?
- How to choose the correct membrane for your specific western blotting needs
Why do I need a membrane anyway?
A wide range of solid phases are available for immobilization of proteins after gel elctrophoresis, ranging from the truly solid phase such as glass or plastic to latex and cellulose that are porous. The most common phases used for blotting comprise microporous surfaces and membranes such as nitrocellulose and polyvinylidene fluoride (PVDF). Microporous surfaces have unique properties that make them ideal for Western Blots.
These properties include:
- Large volume-to-surface area ratio
- High binding capacity
- Storage of immobilized proteins
- Easily allow a solution phase(your antibody) to interact with the immobilized protein
- Lack of interference with detection reagents
For a western blot, microporous surfaces are used in the form of membranes with an average thickness of 100 microns and an average pore size that ranges from 0.05 to 10 microns in diameter. The specific interaction between these membranes and proteins is still not completely understood.
Polyvinylidene fluoride (PVDF) and nitrocellulose are the two membrane types most commonlyused in Western blotting applications.
Nitrocellulose western blot membrane, what is it and why should I use it?
Nitrocellulose is perhaps the most versatile of all the surfaces for the immobilization of proteins. This unique polymer derived from cellulose has been used as the most common immobilization surface in biological research for over 65 years. Even though the exact mechanism by which biomolecules interact with nitrocellulose is unknown, several lines of evidence suggest that the interaction is noncovalent and hydrophobic.
Nitrocellulose is unique, when compared with other microporous membranes, in its ability to easily distinguish between small and large proteins and complexed versus uncomplexed molecules. Nitrocellulose exhibits the highest sensitivity with very low backgrounds in all transfers, especially in protein blotting.
It can be stained with many different protein stains including Coomassie Blue and Ponceau S. The background staining tends to be higher with CBB while Ponceau S gives a very clean pattern but with slightly less sensitivity.
Nitrocellulose wets out naturally, does not require methanol, and will not turn hydrophobic during the transfer process. Nitrocellulose is very easily blocked and does not need the many blocking steps required with PVDF.
Another change to note is that the SDS tolerances are not equivalent for PVDF and Nitrocellulose. The binding of protein to PVDF is much more sensitive to SDS levels. Too much SDS can inhibit the protein’s ability to bind to the PVDF and can, in fact, help proteins already bound to the membrane to slip off. SDS levels should never exceed 0.05% for PVDF.
One clear disadvantage of nitrocellulose is the fact that it cannot be stripped and reprobed multiple times owing to its fragile nature. It also has a tendency to become brittle when dry. In addition, small proteins tend to move through nitrocellulose membranes and only a small fraction of the total amount actually binds.
PVDF western blot membrane, what is it and why should I use it?
Polyvinylidene difluoride (PVDF) is a linear polymer. Proteins transferred PVDF during western transfer are retained well on the membrane surface throughout the immunodetection process via a combination of dipole and hydrophobic interactions. The antigen binding capacity of the membrane is 170 μg/cm2 for bovine serum albumin and this is proportionate with the binding capacity of nitrocellulose. In addition, the PVDF membrane has very good mechanical strength and like Teflon(a related fluorocarbon polymer) it is compatible with a range of chemicals and organic solvents.
Blotting mechanics are not different from those seen with nitrocellulose, except that it is necessary to pre-wet the membrane in either methanol or ethanol before using with aqueous buffers. This is because PVDF is highly hydrophobic and there is no added surfactant in PVDF.
One of the advantages of electroblotting proteins onto PVDF membranes is that replicate lanes from a single gel can be used for various purposes. Proteins blotted to PVDF membranes can be stained with amido black, India ink, silver nitrate, and Coomassie Blue. PVDF membranes offer better protein retention, physical strength and broad chemical compatibility. The higher mechanical strength and superior chemical resistance of PVDF membranes make them ideal for a variety of staining applications and reprobing in immunodetection. Typical binding capacity of commercially available nitrocellulose membranes is 80–100 µg/cm2 while PVDF membranes offer a binding capacity of 100–200 µg/cm2.
A take-home message
Use the membrane that is right for your western blot, and don’t be afraid to change membrane types. Membrane choice depends on a large number of factors including protein size and end point detection. Don’t get locked into a membrane type cause “that’s what my lab has always used.” The answer to your perfect western blot may lay in a change of membranes!
Regardless of the type of membrane used, exceeding the protein binding capacity of the membrane reduces the signal obtained in immunoblotting. Excess protein, weakly associated with the membrane, is readily accessible to react with the primary. However, the resulting antibody–protein complexes will easily wash off during further processing of the membrane.
Good luck and Happy Western blotting!