PVDF vs Nitrocellulose Membrane in Western Blot – Which One to Choose?

Western blotting can be tricky sometimes — protein charge, hydrophobicity, buffer choices, and non-specific binding of other proteins all have a way of making things complicated. But one thing that doesn't always get enough attention is choosing the right membrane.

Most of us end up choosing between two main players: PVDF and nitrocellulose (NC) membranes. In this article, we’re going to break down what makes them different, where to use them for different applications, and how you can pick the best one for your experiment without a ton of trial and error.

PVDF Membranes

The PVDF (Polyvinylidene Fluoride) transfer membranes are naturally hydrophobic. Proteins bind to PVDF membranes through dipole and hydrophobic interactions. PVDF tranfer membranes are more suitable for detecting high molecular weight proteins.

Advantages

High Protein Binding Capacity: PVDF membranes have a high protein binding capacity of 150-300 μg/cm², which also means high sensitivity. This feature makes them a good choice for detecting low expressed proteins.

Durability: PVDF membranes are tough. They handle harsh chemicals without cracking, curling, or falling apart. Its high chemical resistance works well with common stains such as Colloidal Gold, Coomassie Blue, and Ponceau-S. Additionally, their strong hydrophobicity enhances protein retention, reducing protein loss after binding. These features make PVDF membranes ideal for repeated stripping and re-probing.

Stripping a Western blot is a method of removing primary and secondary antibodies from the membrane, allowing it to be re-probed. In theory, a blot can be stripped and re-probed multiple times to visualize different proteins without running a whole new gel. Stripping and re-probing are typically performed when analyzing multiple proteins with different antibodies or when re-evaluating unexpected results.

Disadvantages

Background Noise: The high sensitivity of PVDF membrane can sometimes backfire. If you're working with high expressed proteins, you might end up with more non-specific binding and background noise than you want.

Pre-treatment: PVDF membranes need a little extra prep work. You have to "activate" them by soaking them in methanol (or ethanol) before you use them for protein transfer. After that, you can use them with a methanol-free buffer.

Autofluorescence: If you're doing chemiluminescence-based Westerns, both PVDF and nitrocellulose membranes work fine. But for fluorescent Western blotting, the regular PVDF membrane’s autofluorescence may lead to high background noise.

If you’re planning a fluorescent detection, you should choose low-fluorescence PVDF membranes. Besides, it’s a good idea to cut a small piece of the PVDF membrane, image it both wet and dry, and check the background before using it.

NC membranes

NC (nitrocellulose) membranes are naturally hydrophilic. Proteins are believed to bind to them mainly through non-covalent and hydrophobic interactions. High concentrations of salt and low levels of methanol can improve the immobilization efficiency of NC membranes. Nitrocellulose is usually a better choice when you're detecting medium to low molecular weight proteins.

Advantages

Low Background Noise: Nitrocellulose membranes have a protein-binding capacity of about 80–100 μg/cm², which is lower than PVDF. The lower sensitivity of NC membranes works well when detecting high-abundance proteins because it helps reduce background noise. Plus, NC membranes tend to be more affordable than PVDF, making them a good option for more applications.

Ready to use: Nitrocellulose membranes do not require pre-treatment like PVDF (DO NOT wet the membrane with methanol, or it will dissolve). However, you do need some methanol in your transfer buffer. This is because methanol helps strip SDS from masking the proteins, allowing proteins to bind more effectively to the NC membrane.

That said, methanol may reduce the pore size of the gel, alter protein charge, and cause the precipitation of large proteins. It’s a good idea to test the methanol concentration based on your experimental conditions. Some protocols choose to replace 20% methanol with 10% ethanol in the transfer buffer.

Disadvantages

Fragility: Nitrocellulose membrane is mechanically weak and brittle, which makes it difficult to strip and re-probe without losing signal during antibody washing. 

If you need to strip and re-probe your membrane, it’s better to go with a supported nitrocellulose membrane. Supported versions are more durable and resilient than standard ones.

PVDF vs. Nitrocellulose Comparison Table

Feature	PVDF Membrane	Nitrocellulose Membrane
Protein Binding Capacity	150–300 µg/cm²	80–100 µg/cm²
Protein Size	Better for high MW proteins	Better for mid to low MW proteins
Hydrophilic / hydrophobic	hydrophobic	hydrophilic
Durability	High	Low
Chemical Resistance	High	Low
Background Noise	Can be high	Low
Pre-Wetting Requirement	Requires methanol or ethanol pre-wetting	Requires methanol in transfer buffer
Strip & Re-probe	Performs well	Not recommend, can lose signal
Autofluorescence	High for standard PVDF membrane	Low
Detection	Chemiluminescence detection. For fluorescence detection, better choose dedicated low-fluorescence PVDF membranes.	Both Chemiluminescence detection and Fluorescence detection
Cost	High	Low

Choosing the Right Transfer Membrane for Your Experiment

Here is a quick guide to help you choose the right membrane.

When to Choose PVDF transfer membrane:

Detecting high molecular weight proteins: PVDF membrane shows better binding ability to high molecular weight proteins.

Detecting low-abundance proteins: PVDF membrane’s high binding capacity makes it ideal for detecting low expressed proteins.

Multiple stripping and re-probing: PVDF membrane has greater durability and higher chemical resistance than NC membrane. Its high protein binding capacity also helps retain proteins during the stripping and re-probing process.

When to choose Nitrocellulose transfer membrane:

Detecting mid to low molecular weight proteins: NC membrane has better binding efficiency to low molecular weight proteins than PVDF membrane.

Cost-effectiveness: Nitrocellulose transfer membrane is generally cheap and meets most experiment requirements. If you are looking for a cost-effective and easy-to-use transfer membrane (No need for pre-wetting) , it is a good choice.

Minimizing background noise: If minimizing background noise is the priority consideration, the nitrocellulose transfer membrane may be the best choice. 

About Pore Size

Both PVDF and Nitrocellulose membranes are available in 0.2μm and 0.45μm pore sizes.

0.2μm: Best for detecting low molecular weight proteins. It offers a higher protein-binding capacity and better retention compared to 0.45 μm membranes. It’s especially recommended for proteins smaller than 20 kDa and works well for detecting low-abundance proteins.

0.45μm: Good for general Western blotting. It’s ideal for proteins larger than 20 kDa and typically results in lower background noise.

Conclusion

Both PVDF and nitrocellulose membranes have their advantages in Western Blotting. When choosing a membrane, it's worth considering things like protein size, detection sensitivity, whether you’ll need to strip and re-probe, and of course, your budget.

If you need high sensitivity and plan to strip and re-probe multiple times, PVDF is probably the better pick. But if you’re after a cost-effective, easy-to-use membrane with minimal background noise, nitrocellulose is a solid choice.