Nitrocellulose Membrane for Western Blot

What is a nitrocellulose membrane?

Nitrocellulose membranes are one of the top matrices used in protein blotting. They have high protein-binding affinity, compatibility with a variety of detection methods, and the ability to immobilize proteins, glycoproteins, or nucleic acids. Examples of compatible detection methods include chemiluminescence, chromogenic, and fluorescence. It is proven to produce excellent signal-to-noise results when used for amino acid analysis and western, northern, and Southern blotting.

What is a western blot?

Western blotting (also called Protein Immunoblotting) is an analytical technique used to detect specific proteins in the given sample. It uses SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in the sample. The separated proteins are then transferred or blotted onto a matrix, where they are stained with antibodies specific to the target protein. Expression details of the target proteins in the given cells or tissue homogenate can then be obtained through analyzing the location and intensity of the specific reaction. Western blotting analysis can detect target protein as low as 1 ng due to high resolution of the gel electrophoresis and strong specificity and high sensitivity of the immunoassay. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines for various experiments.

How do proteins bind to nitrocellulose membranes?

It is believed that protein immobilization is the result of hydrophobic interactions. Nitrocellulose membranes have a non-hydrophobic nature, allowing the proteins to bind to them through electrostatic interactions. This is enhanced with high salt and low methanol concentrations, which assist with protein immobilization during the electrophoretic transfer, especially for proteins with higher molecular weights. However, nitrocellulose membranes are not optimal for the electrophoretic transfer of nucleic acids, as the high salt concentrations required for efficient binding will eliminate some or all of the charged nucleic acid fragments. Nitrocellulose membranes will also readily dissolve in organic solvents such as acetone and methanol, requiring low concentrations of the latter to prevent disruption. Nevertheless, nitrocellulose membranes can still provide substantial results when used with nucleic acid.

What are the options for western blot transfer membranes?

The three main types of transfer membranes are nitrocellulose, polyvinylidene fluoride (PVDF), and nylon (polyamide). The pros and cons and differences are described in the following table:

PVDF Nitrocellulose Nylon
Protein size Better with high molecular weight proteins Better with mid to low molecular weight proteins (< 20 kDa) Better with nucleic acids
Binding capacity 100 – 200 μg/cm2 80 – 100 μg/cm2 > 400 μg/cm2
Background noise Slightly low Very low High
Strip and re-probe Performs well Possible but may lose sensitivity Performs well
Mechanical strength High Brittle when dry High
Solvent resistance Strong Weak weak
Pre-treatment Wet with methanol Wet with buffer Wet with buffer
Pore size 0.2 μm and 0.45 μm options 0.2 μm and 0.45 μm options 0.45 μm

One of the greatest benefits of using the nitrocellulose membrane over the other membranes is the very low background noise. The background is the darkening of the blot as the protein travels through the membrane. A high background noise equates to a highly darkened blot, which means it is difficult to distinguish specific bands. The very low background noise seen with the nitrocellulose membranes helps to ensure the experiment can be run with minimal difficulty interpreting the results.

Where can I purchase nitrocellulose membranes?

Boster Bio specializes in manufacturing primary antibodies for WB, IHC, and ICC/IF as well as producing high quality ELISA and reagent kits. Our lab runs hundreds of WB every week for our internal production quality control, and we use the reagents and kits we manufacture ourselves for our ELISA kits and IHC- and WB-validated antibodies, so we ensure all our extraction kits, buffers, controls, solutions, etc. will help you obtain clean and specific results for accurate localization and identification of antigens.

We provide nitrocellulose membrane for western blot transfer with both pore 0.2 μm (Catalog# AR0135-02) and pore 0.45 μm (Catalog# AR0135-04) options. Boster’s nitrocellulose membranes can be used for a wide range of protein molecular weights and nucleic acids > 500 base pairs in size. Each ordered pack contains 20 sheets measuring 9 cm x 10 cm in size. They can remain stable for one year when stored at room temperature. The products are high quality, pure 100% nitrocellulose membranes with a high surface area and excellent uniformity. The variety optimizes for peptide applications with the 0.2 μm pore size and protein applications with the 0.45 μm pore size.

How can nitrocellulose membranes be used in western blots?

There are five main steps involved in the western blot workflow, described as follows:

  • Transfer

    : Proteins are moved from within the gel onto the nitrocellulose membrane. Without pre-activation, proteins combine with the membrane based on hydrophobic interaction. Therefore, there is only a slight effect on the protein activities.

  • Blocking

    : In a western blot, it is important to block the unreacted sites on the membrane to reduce the amount of nonspecific binding of proteins during subsequent steps in the assay using inert protein or nonionic detergent. The chosen blocking buffers should block all unreacted sites, should not replace target protein on the membrane, not bind epitope on the target protein, and not cross react with antibody or detection reagents.

  • Primary Antibody Incubation

    : After blocking, a primary antibody specific to the target protein is incubated with the nitrocellulose membrane. This allows the primary antibody to bind to target the protein on the membrane.

  • Secondary Antibody Incubation

    : After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to a specific enzyme-conjugated secondary antibody. The chosen secondary antibody will bind to the primary antibody, which has already reacted with the target protein.

  • Western Blotting Analysis

    : After color development, the pattern of the separated proteins is imprinted onto a film or captured by Western Blot gel imager. This allows for comparison of the band position to the protein ladder, thereby estimating the size of the protein.

References

  • Kurien, B. T. and Scofield, R. H. (2015). Western blotting: an introduction. Methods Mol Biol, 1312, 17-30.
  • Xiang, Y., Zheng, Y., Liu, S. et al. (2021). Comparison of the sensitivity of Western blotting between PVDF and NC membranes. Sci Rep, 11, 12022.
  • Xu, J., Sun, H., Huang, G. et al. (2019). A fixation method for the optimisation of western blotting. Sci Rep, 9, 6649.

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