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.
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.
Polyvinylidene difluoride (PVDF)
PVDF membranes on the other hand are a bit more complex to work with, but also more robust. 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.
While most labs use PVDF or nitrocellulose membranes other options are available include nylon or cellulose membranes. However, these are usually used only in specific circumstances and so we will not cover them here.
Which one is right for me
Well 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:
- Repeated re-probes – PVDF
- Small proteins <25 kDa – Nitrocellulose
- Hydrophobic protein – Nitrocellulose
- Low abundance – PVDF
- High background signal – Nitrocellulose
Hopefully the above tips will help you pick the right membrane for your assay, but it’s always worth considering a change if you’re not getting the bands you expect.