While we might not like to admit it, us scientists are creatures of habit, reducing variables to ensure our hypothesis testing is as accurate as possible rapidly creeps into our day to day lab tasks. In some ways this can be good, with standard operating procedures and recipes being handed down through the lab to ensure consistency, but in other ways it can be limiting. Today I’m going to talk about one area that’s particularly close to my heart… Western blotting membrane choice.
Now I’ll freely admit it’s not the coolest of topics, but it’s something worth considering, especially if you’re struggling with your Western blotting. I went through a period of using exclusively polyvinylidene difluoride (PVDF) membranes as that was what I’d always used. One protein though was giving me a lot of trouble, lots of background regardless of antibody used, block or transfer conditions. At the end of my tether and tearing my hair out I borrowed some nitrocellulose membrane as a last resort, and it worked straight away. So sometimes being flexible can be beneficial as well.
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How to choose the right membrane for Western blotting
In the majority of situations, either PVDF or nitrocellulose membranes will probably work well for you, but there are certain circumstances when one is preferred over the other. Choosing between membranes depends on your detection method, whether you intend to strip and reprobe, and your target protein(s) (Table 1).
While you can use both nitrocellulose and PVDF membranes for your Western blot analysis, nitrocellulose membranes are ideal for detecting low molecular weight proteins and PVDF membranes are more suitable for detecting higher molecular weight proteins. Both PVDF and nitrocellulose membranes are compatible with chemiluminescence-based Western blotting. For fluorescence-based Western blot detection, we do not recommend using a nitrocellulose membrane, due to its high autofluorescence.
Two most common types of membranes used in Western blotting
Sometimes your experiment may require a more sophisticated membrane than standard. Using the optimal membrane for your Western Blot application can be critical to your experiment’s success.
Table 1. How to choose between nitrocellulose and PVDF membranes for Western blotting
| Nitrocellulose | PVDF | |
|---|---|---|
| Repeated re-probes | Appropriate | Possible, but sensitivity is lost |
| Small proteins | <25 kDa | >25 kDa |
| Detection | Great to use for chemiluminescent and fluorescent detection | Use dedicated low-fluorescence PVDF membranes for chemiluminescence detection |
| Protein binding | Hydrophobic proteins | Hydrophobic and dipole proteins |
| Sample concentration | Low abundance 80-100 µg of protein/cm2 | Higher abundance 150-200 µg of protein/cm2 |
| Autofluorescence | Low | High with standard PVDF membranes |
Nitrocellulose Membranes
Nitrocellulose was the first Western blot membrane and is still widely used today. Ready to use and easily hydrated nitrocellulose membranes rapidly and readily bind with proteins out of the box and are often more affordable than other membrane types. However, they often lack durability making stripping and re-probing more difficult.
Why are nitrocellulose membranes used in Western blotting?
Nitrocellulose membranes are extensively utilized in Western blotting due to their superior protein-binding affinity, compatibility with multiple detection methods (chemiluminescence and fluorescence, to name a few), and efficacy in immobilizing proteins, glycoproteins, or nucleic acids.
Polyvinylidene difluoride (PVDF) Membranes
PVDF membranes are more robust, but a bit more complex to work with. They need to be activated in methanol before they can efficiently bind proteins, and often also require methanol and SDS in the transfer buffer.
Why are PVDF membranes used in Western blotting?
PVDF membranes are utilized because they are robust and can bind more protein than their nitrocellulose counterpart. They are ideal for low abundance proteins. PVDF membranes offer increased sensitivity, with the tradeoff of being more prone to showing non-specific background staining.
Other types of membranes
While most labs use PVDF or nitrocellulose membranes, other options are available. These include nylon or cellulose membranes. However, these are usually used only in specific circumstances. I hope after reading these tips we were able to help you pick the right membrane for your assay. It’s always worth considering a change if you’re not getting the bands you expect. Make sure you have everything you need for your next Western blot, by checking out this Western blot resources list.
If you’re still having issues selecting the right membrane, fill out the form on this page and one of our scientists will help you narrow it down to the right membrane.
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