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, and 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.
How to choose which membrane is right for Western blotting
In the majority of situations, both membranes will probably work well for you, but there are certain circumstances when one is preferred over the other. Choosing between nitrocellulose and PVDF 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 recommend using a nitrocellulose membrane, due to its high autofluorescence.
|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
150-200 µg of protein/cm2
|Autofluorescence||Low||High with standard PVDF membranes|
Table 1. How to choose between nitrocellulose and PVDF membranes for 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.
Two most common types of membranes used in Western blotting
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.
Azure offers multiple sizes of nitrocellulose membranes to accommodate larger and smaller proteins. Each package comes with ten membranes in two pore sizes: 0.45 μm and 0.22 μm.
Polyvinylidene difluoride (PVDF) Membranes
PVDF membranes are more robust, but a bit more complex to work with. Requiring activation in methanol before they can efficiently bind proteins, and often requiring methanol and SDS in the transfer buffer PVDF membranes bind more protein than nitrocellulose making them ideal for low abundance proteins. However, because of their increased sensitivity they are more prone to showing non-specific background staining.
Pre-cut PVDF membranes can be used for a variety of Western blotting applications to save you time in the lab. Consider using the low-fluorescence PVDF membranes from Azure to reduce background noise for improved sensitivity.
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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More posts on Western blotting:
Shop Membranes and Reagents for Western Blotting
Azure Fluorescent Blot Blocking Buffer
Azure Protein-free Blot Blocking Buffer
Azure Chemi Blot Blocking Buffer