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.
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.
Multiple sizes of nitrocellulose membranes are available on the market to accommodate larger and smaller proteins. Azure offers packages of ten membranes in two pore sizes, 0.45 μm and 0.22 μm.
Polyvinylidene difluoride (PVDF)
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 and so we will not cover them here.
Which membrane is right for me?
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:
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.