Choose the Right Western Blot Membrane for Your Experiment

How to Select the Best Western Blot Membrane

Tuesday, 21 January, 2020


western blot membrane,nitrocellulose,PVDF,choosing a blotting membrane

Choosing the right membrane for your experiment is target-dependent and therefore extremely important. There are two different types of membranes that you can use in protein transfer: nitrocellulose and polyvinylidene fluoride (PVDF). Both membranes work best under specific experimental conditions. Before continuing with your research, make sure that you understand their respective advantages and disadvantages.

Nitrocellulose membrane

Nitrocellulose membranes typically have a lower protein binding capacity (80-100 µg of protein/cm2) than PVDF membranes. Pore sizes can be either 0.2 µm or 0.45 µm. The 0.45 µm pore membrane is used for most targets; because the 0.2 membrane is more efficient in binding smaller proteins, however, you can switch to it when probing for ones that are less than 15 kDa. Additionally, transfer buffer must contain methanol, and it is important to note that nitrocellulose membranes can be fragile. Nitrocellulose is well suited for both chemiluminescence and fluorescence detection methods.

Polyvinylidene fluoride (PVDF)

Compared to nitrocellulose membranes, PVDF has a higher protein binding capacity (150-200 µg of protein/cm2). However, both membranes are comparable in pore size (0.2 µm or 0.45 µm). PVDF does not necessarily require a transfer buffer with methanol, but it must be pre-wetted with it. The PVDF membrane is less fragile and is consequently recommended for experiments that utilize stripping and re-probing of the membrane.

Detection

Figure 1. Western blot detection of transferrin using various vendors and brands of PVDF membrane. PVDF membranes were scanned on the Odyssey Classic Infrared Imaging System in both 700 and 800 nm channels. LI-COR offers prescreened Immobilon-FL PVDF membranes in convenient, affordable kits.

While it works well for chemiluminescence and fluorescence detection methods, you should select low-fluorescence PVDF membranes when performing fluorescent Western blots. This will help you avoid high background due to the autofluorescence of standard PVDF membranes. To check for autofluorescence and background, cut a small membrane sample and image it both wet and dry.

How can your Western blot performance be affected by your choice of membrane and other variables? Find out in the “Good Westerns Gone Bad: Tips to Make NIR Western Blots Great” application guide.

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