PVDF vs Nitrocellulose

Overview:

Every researcher aims to conduct an experiment in which all conditions are suitable for the purpose of the investigation. However, there are always those certain elements to scientific protocols that are specifically tailored to precise circumstances, especially in well-established procedures such as Western Blotting.

Western Blotting is a very precise experimental technique which also has many varying dilutions, reagents, timings for certain equipment and above all, transfer membrane type.

Here we will be looking at the two most common options for transfer membrane which can be used when performing a Western Blot; Polyvinylidene Difluoride (PVDF) Membrane and Nitrocellulose (NC) Membrane.

PVDF and Nitrocellulose have their own unique qualities as a transfer medium, which can benefit one set of conditions in an experiment over the other.

In order to help you make a decision between which membrane is best for your research, we will explore the differences between PVDF Membrane and Nitrocellulose Membrane.

Pore size:

A key factor in choosing your membrane is going to be its pore size. Both PVDF and Nitrocellulose are produced with multiple pore size options, with the pore dimensions dictating the ability of protein migration between different sized proteins.

0.45um pore size is considered the most common size on the market, but as protein molecular weight dips below the 15kDa mark, a transfer medium with a 0.2um pore size may be more suitable. It is also worth considering that if the loading volume of your target protein is relatively low, and/or quantification of said protein is a key element in your research, then it’s wise to choose a membrane with a smaller pore size.

Transfer buffer:

Your choice in transfer membrane will also determine the relevance and importance of Methanol in your research protocol. With Nitrocellulose (NC) it does not require methanol for activation, but it is essential for methanol to be present within the transfer buffer.

This is because it helps strip SDS from masking the proteins migration ability as well as the precipitation of high molecular weight proteins.PVDF on the other hand, the membrane MUST be soaked in methanol for activation before it can be used as a transfer medium.

When using PVDF, the transfer buffer does not require methanol as it can be a useful technique for high molecular weight proteins that precipitate in low SDS/high methanol solutions.

Reprobing and detection:

If you wish to re-probe your membrane after completing your experiment, it is best to know that PVDF is the better choice as Nitrocellulose is innately fragile, making it quite difficult to re-probe on without losing the signal during the washing of antibodies.

PVDF membrane is notably more durable and PVDF has a much better retention of absorbed proteins due to its hydrophobicity. This allows membranes to be stripped and re-probed with ease.

Both PVDF and Nitrocellulose are well suited for experiment protocols like western blotting, chemiluminescence and fluorescence detection methods. However, in the circumstances where you are conducting fluorescence detection with PVDF, low-fluorescence must be used in order to avoid high background.

A high background can be the result of autofluorescence from standard PVDF membranes. It is recommended that you cut a small sample of membrane and image it both wet and dry, to check for autofluorescence and background.

Summary:

When comparing the two western blot membranes overall, it is worth noting that if your protocol will involve small sized proteins, below 20kDa in weight, or you wish to re-probe your membrane after conducting a western blot, then it would be best to consider the specificity between membranes previously outlined. Nitrocellulose membrane has a high affinity for proteins and has a high retention for generating a signal and is typically cheaper than PVDF membrane.

However, PVDF has the ability of re-probing and storage of data. These are things Nitrocellulose is unable to deliver towards research investigations. Even when considering modified Nitrocellulose membranes designed for re-probing, having to purchase this over regular Nitrocellulose is rather expensive.

A fault to PVDF membrane is that it can often produce a harsh background, so extra care is needed when washing the membrane. Nitrocellulose, on the other hand, requires the use of methanol in transfer buffers, this is often used as a way to remove SDS. The presence of methanol can risk reducing the pore size of the gel, which can restrict the transfer of some molecules.

With each of the two transfer membranes possessing their own separate pros and cons, the overall take when choosing between Nitrocellulose or PVDF is that it can be down to personal preference as well as scientific relevancy.