Western blotting is a staple procedure in most labs worldwide and has become the gold standard for protein profiling and quantification. The principle of the Western blot is to take a complex protein mixture that has been separated in a gel (by molecular weight or charge) and transfer it onto a membrane, where it will be probed with an antibody for identification and quantification.
Choosing the right membrane is a key factor in successful Western blotting. You want to select a membrane that will deliver signal while resisting background and nonspecific binding. When making this critical decision for your application, focus on the two main factors that will affect your experiment’s outcome: membrane type and pore size. After you’ve decided on these two factors, you’ll also want to consider which format best suits your application.
The top types: Weighing the pros and cons
Let’s first look at the most common membranes used in Western blotting: polyvinylidene difluoride (PVDF) and nitrocellulose. Each offers key attributes to suit particular experimental conditions. To produce good Western blot data, it’s vital to evaluate the physical properties and performance characteristics of each membrane type.
PVDF for durable membranes and high sensitivity. PVDF membranes are a popular choice for Western blotting applications because of several key characteristics. First, they offer high mechanical strength and chemical resistance, so they’re durable for reprobing. PVDF membranes also typically have higher binding capacities than nitrocellulose—150 to160 µg/cm2 vs. 80 µg/cm2—which means they offer higher sensitivities.
Because of the hydrophobicity of PVDF, these membranes are also the preferred choice for hydrophobic (i.e., membrane) proteins. But consequently, this membrane requires a brief “wetting” step with methanol. If you’re selecting PVDF for a fluorescent blot, choose a low-fluorescent PVDF membrane, which offers the same advantages as a standard PVDF membrane but with minimal autofluorescence.
Nitrocellulose for fast binding and steady support. Nitrocellulose was one of the first membranes used for Western blotting and remains a popular choice, as protein binding to nitrocellulose is instantaneous, nearly irreversible and the membrane is easily hydrated.
Traditional nitrocellulose membranes are not suitable for stripping and reprobing or harsh chemical treatments. For such situations, supported nitrocellulose is a better option. Supported nitrocellulose contains an inert support structure, giving the membrane increased strength and resilience.
4000x magnification of PVDF (A) and nitrocellulose (B) membranes, showing structural differences.
Use pore size to prevent escape
The second consideration for choosing the right membrane for your application is membrane pore size. Western blotting membranes are microporous substrates to which proteins bind; the dimensions of the pore dictate the size of the protein that can successfully bind to the membrane without passing through.
Western blotting membranes are commonly available in two pore sizes. You have 0.45-µm pore membranes for most analytical blotting experiments, in which proteins have a molecular weight greater than 20 kD. Or there are the smaller 0.2-µm pore membranes, which are most suitable for low molecular weight proteins (less than 15 kD) that might move through larger pores. A good rule to follow: If detecting a protein loaded at low levels, or quantification is critical, choose the smaller size membrane.
Membrane formats
Western blot membranes are packaged in numerous formats. When selecting the format, you must consider your transfer system (semi-dry, wet or fast), convenience, price and flexibility. The formats currently available on the market are discussed below.
Rolls. These are a very popular choice, as rolls comprise a large area of membrane (e.g., 26 cm x 3.3 m). You can cut the membrane to the specific size of your gel, which offers more flexibility. However, this can add extra time to your workflow and introduce variability in the membrane size. It is important that the size of the membrane corresponds to the size of the gel, so you maximize your transfer efficiency.
Precut membranes. These membranes are precut by the manufacturer, which is a great time saver, and they are available in a range of sizes for all gel types. Using a precut membrane may result in better transfer reproducibility.
Precut and pre-wetted membranes. If convenience, reproducibility and high throughput are important to you, then these are the best membranes to choose. Transfer efficiency not only relies on choosing the right membrane, you also have to make sure the buffer promotes a quick and even transfer.
An informed choice will pay off
There is a lot of uncertainty surrounding Western blotting, as it is difficult to control the experiment parameters. Making the right decisions during experimental design can help you have greater confidence in your final results.
It is important to validate the correct membrane type for your experimental conditions. Choosing the correct membrane type for Western blotting can help prevent transfer inefficiencies, reduce background after staining and increase the accuracy of your protein quantification. Consider your options wisely and reap the reward of reliable results.
Thomas Davies is the global product manager for Bio-Rad Laboratories' Lab Separation Division. He earned his degree in biotechnology from the University of Hertfordshire England, working on creating saccharomyces models for apoptosis. Davies has worked for a number of high-profile biotech companies in technical support and sales-specialist positions supporting proteomics products. Kenneth Oh, Ph.D., is a senior R&D scientist for Bio-Rad Laboratories Inc. Oh earned his degree in biochemistry from the University of California, Santa Barbara. There, he characterized peptide-protein and peptide-nucleic acid interactions by synthesizing unique fluorophores and monitoring their conformational-dependent steady-state and kinetic fluorescence properties. Oh was the co-founder of Chimeros, where he synthesized viral, capsid-like nanoparticles that served as delivery vesicles for RNAi therapeutics.
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