Western blotting is an essential technique used in labs and industries to separate and identify proteins. The procedure starts by separating proteins based on molecular weight through gel electrophoresis, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the separated proteins are transferred to a membrane followed by their visualization using a suitable detection method.
The overall process is divided into six steps:
NOTE: It’s recommended to perform the washing and antibody incubation at room temperature to ensure even agitation.
The two types of membranes commonly used in Western blotting assay for protein transfer are nitrocellulose and Polyvinyl difluoride (PVDF) membranes. Both these membranes have a similar appearance, yet, they differ significantly in their properties.
In this article, we will cover more about both types of Western blotting membranes and some factors you need to consider before making a choice for the right membrane for your application.
A successful protein analysis workflow depends on Western blot membranes. The two types of membranes extensively used in labs include nitrocellulose and PVDF membranes.
They both come with a pore size of 0.1, 0.2, and 0.45 μm. The membrane blots of 0.45 μm are required for protein blotting applications whereas 0.1 and 0.2 μm are required for quantitative and qualitative analysis of smaller peptides or proteins with molecular weight less than 15 kDa.
Before making a decision on the uses of the membranes for your workflow, it’s crucial to understand their advantages and disadvantages.
Figure: Electron microscopy images of PVDF membranes (Left side) and nitrocellulose membrane (Right side).
Nitrocellulose membranes are one of the oldest cellulose derivatives. These proteins are immobilized on membrane surfaces through hydrophobic interactions, which can be enhanced by using low methanol and high salt concentrations.
Nitrocellulose membranes are frequently used as solid support in protein and immunoblotting techniques such as Southern, Northern, and Western blotting for the detection, quantitation, or quantification of biomolecules. This is mainly due to the following properties:
The protein binding capacity of nitrocellulose membrane ranges from 80 to 100 µg/cm2. Therefore, it’s considered to have lower sensitivity than Polyvinylidene fluoride membranes to detect proteins with low expression. Additionally, due to their brittle nature, you need to be careful while stripping or reprobing nitrocellulose membranes to prevent signal loss.
The advantage of these membranes is that they have lower background signals and can be used to detect low molecular weight proteins.
Nitrocellulose membranes with different pore sizes are available in the market. For example, 0.2 µm pore size is mainly used to transfer nucleic acid and low molecular weight proteins. Thus, it’s essential to understand the membrane of which pore size will be suitable for your applications.
PVDF membranes are extensively used in blotting methods for the electrophoretic transfer of proteins from a gel onto the membrane. The membranes are used for downstream applications, such as protein sequencing and immunodetection.
PVDF membranes have a binding capacity of 170 to 200 µg/cm2. Thus, it offers more sensitivity for quantitative analysis or estimating the expression of specific proteins with low abundance. Furthermore, the transfer membrane is ideal for sequencing and reprobing.
PVDF has been a part of protein blotting techniques for a long time due to its outstanding properties, such as:
The drawback of PVDF membranes is their high background noise caused by autofluorescence.
Western blot membranes are extensively used in immunoblotting and protein analysis experiments. They act as solid-phase support for the proteins and do not react with antibodies, providing better analysis of biomolecules.
After the proteins are separated in a gel based on their molecular weights, they are transferred to a blotting membrane using an electric field. Before the transfer, the membrane activation is done by soaking it in methanol.
During the transfer procedure, the membrane and gels are placed together with filter papers on both sides, between two electrodes. Then, the current is passed across the gel to the membrane for the transfer of proteins.
Figure: Gel and membrane setup for transfer of proteins.
Today, precut and pre-assembled membranes and filter papers are available that perfectly fit your gels and make the process easier and faster.
The reagents used to transfer proteins include wet or semi-dry transfer buffer, which consists of tris base, glycine, methanol (concentration is based on the type of buffer prepares for the assay), TBST (Tris-buffered saline (TBS) with 0.05% Tween-20) wash buffer, blocking buffer (5% bovine serum albumin (BSA), and others.
The electron microscopy of a Western blotting membrane shows random, interconnected pores with a sponge-like appearance. This pore size can be altered during manufacturing for the analysis of biomolecules of different sizes.
Though nitrocellulose and PVDF membranes look similar, they have many differences. Therefore, before choosing the right membrane for your assay, consider the following factors:
If you’re working with non-abundant proteins, then PVDF membranes would be a suitable choice for your workflow because of their higher sensitivity and binding capacity. However, if you’re willing to study the high level of protein expression, then you may prefer the nitrocellulose membrane that offers lower background noise.
Both the processes, stripping and reprobing, involve harsh conditions that disrupt the membrane-bound protein’s interaction with the primary antibody. Therefore, PVDF membranes are the most preferred to go through such processes for their durability and chemical-resistant materials, unlike nitrocellulose membrane, which is brittle and fragile.
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Western blot membranes are microporous membranes that offer solid support for the electrophoretic transfer of proteins. It’s a routine workflow in life sciences, medical, therapeutic, and diagnostic labs.
The two most common types used in blotting applications include nitrocellulose and PVDF membrane. Though both membranes look similar in electron micrographs, they have significant differences in their properties. That’s why it’s crucial to consider factors like the target protein in the study, the requirement of stripping and reprobing, and involved detection methods, to decide the right membrane for your application.
For efficient transfer of proteins to the membrane, high-quality reagents are required that provide no background noise or interfere with the assay. Similarly, high-tech, advanced equipment is another essential requirement to obtain reliable, clean, and publishable data. However, both high-quality reagents and expensive equipment contribute to the high cost of lab processes.
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