Immunoblotting as a widely used experimental technique in the fields of biological detection and analysis. The core concept is to transfer the sample to a solid phase carrier and then detect the sample by means of a corresponding detection method. Solid-phase carriers, specifically blot membranes, are widely used in biological detection and analysis, including protein transfer, immunoblotting, and the transfer of traditional DNA and RNA, as well as nucleic acid hybridisation detection.

What are they ?

Blotting membranes are mainly divided into positively charged nylon membranes, nitrocellulose membranes and polyvinylidene fluoride membranes.

·Positively charged nylon(NY⁺) membranes 

Positively charged nylon membranes bind proteins and nucleic acids through ionic, electrostatic, and hydrophobic interactions. They offer high sensitivity, consistent transfer results, and a nucleic acid capacity of 480-600 µg/cm², while a protein binding capacity of 500µg/cm². A notable disadvantage of using nylon membranes for blotting applications is the potential for non-specific binding and strong binding with anions.

Figure 1 NY+ transfer membrane

·Nitrocellulose (NC) membranes

Nitrocellulose membranes are widely used as substrates in protein blotting due to their high protein binding affinity, compatibility with various detection methods, and ability to immobilize proteins and glycoproteins. The protein binding capacity of nitrocellulose membranes ranges from 80 to 100 µg/cm². It is widely accepted that protein immobilization is driven by hydrophobic interactions. It has been demonstrated that high salt concentrations and low methanol concentrations can improve the membrane's ability to immobilise proteins during electrophoretic.

Figure 2 NC transfer membrane

·Polyvinylidene fluoride (PVDF) membranes.

PVDF membranes exhibit a high binding affinity for proteins and nucleic acids. However, due to their highly hydrophobic nature, they must be pre-wetted with methanol or ethanol prior to immersion in transfer buffer. PVDF membranes have a protein binding capacity of 170–200 µg/cm², and their stronger hydrophobicity provides superior protein adsorption and retention capabilities compared to other membranes. In comparison to NC, PVDF membranes are less brittle and fragile, making them suitable for Western Blotting experiments requiring multiple rounds of reprocessing (demarking and remarking steps) using new antibody combinations targeting different targets.

Figure 3 PVDF transfer membrane

How to Choose the Right One？

· NY⁺ membranes

The positively charged nylon membrane has uniformly distributed positive charges on its surface, giving the membrane high sensitivity and low background signal. Therefore, when performing experiments such as Southern, Northern, Dot, and Slot Blot, the positively charged nylon membrane is the preferred choice.

·NC membranes.

In the execution of Southern, Northern, and Western blotting, amino acid analysis, and dot/slot blotting experiments, where cost-effectiveness and ease of use are paramount, NC membranes emerge as a favourable option. However, it should be noted that NC membranes are fragile and cannot be reused.

·PVDF membranes.

Western blotting, amino acid or protein analysis, and other experiments require transfer membranes with high chemical resistance and excellent protein adsorption capacity. PVDF transfer membranes are an ideal choice, as they offer excellent mechanical strength and can be reused. In addition, compared to NC membranes, PVDF transfer membranes offer better solvent resistance and higher signal-to-noise ratios.

Table 1 Comparison of the performance of three common transfer membranes