When it comes to Western blotting, choosing the right membrane for your application usually spells the difference between success and failure. However, this is usually easier said than done since you need to consider the properties of your protein and the downstream detection steps required in your application before you can determine which membrane can give you the results that you need.
You need to choose a membrane that will deliver signal while resisting background and nonspecific binding. So, how can you do this? You should start by trying to learn a little bit more about the membranes that you use in Western blotting.
PVDF and Nitrocellulose: Which Should You Use?
When it comes to Western blotting, most researchers prefer polyvinylidene difluoride (PVDF) and nitrocellulose over other types of membranes since both offers several key attributes that makes them suitable for particular experimental conditions. How does one compare with the other? And how do you choose which one to use in your application? To do this, you need to take the membrane type, pore size and membrane format into consideration to help you choose which membrane to use.
Protein binding capacity. PVDF has a protein binding capacity of 170 to 200 μg/cm2 while nitrocellulose has a protein binding capacity of 80 to 100 μg/cm2. Since PVDF has a higher protein binding capacity, it also offers higher sensitivity. While this feature allows it to detect lowly expressed proteins, you are more likely to get higher background noise in your antibody detection steps when using this membrane. Nitrocellulose membrane, on the other hand, may not be capable of the detection sensitivity of PVDF membranes but it will produce a lower background noise.
Binding interactions. Protein molecules bind to nitrocellulose membranes through hydrophobic interactions while molecules bind to PVDF membranes through hydrophobic and dipole interactions. However, please note that nitrocellulose requires the use of methanol in the transfer buffer which may reduce the pore size of the gel and cause high molecular weight proteins to precipitate.
Physical characteristics. While nitrocellulose is brittle and fragile, PVDF is more durable and has higher chemical resistance making it ideal for reprobing and sequencing applications. Nitrocellulose can prove to be difficult to strip and reprobe without losing signal. However, supported versions of nitrocellulose membranes are considerably more durable and resilient than standard nitrocellulose membranes so they can be stripped, reprobed, and subjected to harsh chemical treatments.
Pore size. Both membranes come in typical pore sizes of 0.1, 0.2 or 0.45μm. The 0.45μm membrane is suitable for most protein blotting applications but for smaller peptides or lower molecular weight proteins (less than 15 kD), you should use 0.1 or 0.2μm pore size membrane. Note: When you are detecting a protein loaded at low levels or when quantification is considered critical, you should always choose the smaller size membrane.
Membrane format. There are several factors that you need to take into account when choosing the most suitable membrane format, including transfer system (semi-dry, wet or fast), convenience, price and flexibility. Pre-cut and pre-wetted membranes are the ideal choice when convenience, reproducibility and high throughput are of highest importance while rolls offers more flexibility since you can cut the membrane to the specific size of your gel. The only problem is that this can add extra time to your workflow and introduce variability in the membrane size. Pre-cut membranes, on the other hand, are available in a range of sizes suitable for all gel types. Using a precut membrane may result in better transfer reproducibility.
While both nitrocellulose and PVDF membranes are used for Western blotting and amino acid analysis, nitrocellulose is ideal in detecting low molecular weight proteins while PVDF is more suitable for detecting higher molecular weight proteins. In addition, nitrocellulose can be used for nucleic acid analysis and dot/slot blotting while PVDF can be used for protein sequencing and solid phase assay systems.
Keep in mind that you can get more reliable results from your Western blot experiment by choosing the correct membrane type. So, choose carefully and you will surely get the results that you need.
Image Source : Robert Couse-Baker
