Using Membranes to Obtain High Sensitivities in Nucleic Acid and Protein Detection

Membranes have been standard tools in the molecular biology laboratory since the 1970’s, when it was discovered that biomolecules could be spotted directly onto membranes (spot ELISA or DNA dot blots) or transferred from gels (Southerns, westerns, and northerns). The first membranes used for molecular detection were made from nitrocellulose, which has high affinity for biomolecules and low background with many detection techniques. Difficulties with handling and exposure to heat led to a search for alternative supports. For nucleic acids, nylon membranes were found to have higher binding and avidity, and the structural integrity to withstand fixation temperatures and the multiple steps involved with detection and reprobing. PVDF membranes were found to have higher affinity for proteins than nitrocellulose, and are favored for western transfers, although nitrocellulose remains in widespread use.

Biomolecules bind to membranes primarily through hydrophobic interactions. Even though a membrane such as nylon may be hydrophilic, hydrophobic domains in the polymer are available to align with hydrophobic domains on the biomolecule (read about a model for binding DNA and protein to transfer membranes). Charge interactions also play a role, allowing highest sensitivity for nucleic acids to be achieved on positively-charged nylon membrane, although the relationship is complex, and is often dependent on interactions with the detection reagents. Similarly, highest levels of protein binding are found on PVDF membranes which offer little opportunity for charge interactions, but are very hydrophobic.

There are many references in literature to the "binding capacity" of different membranes. These specifications are typically greater than 100 µg/cm², or as much as 500 µg/cm², which are far in excess of what can be effectively used for detection. Often, highest signal with immobilized protein or nucleic acid occurs at approximately 1 µg/µL (about 10 µg/cm²), and greater amounts actually result in decreased signal (prozone phenomenon in classic protein terminology). What is important, then, is not the maximum amount of protein that can be loaded onto a membrane surface, but the smallest amount that can be detected. This is related to the membrane’s affinity and avidity, and is commonly expressed as sensitivity.

Membranes with high affinity for biomolecules enable easy immobilization. At the same time, they will also adsorb detection reagents. Early blocking schemes used BSA and gelatin, which were found to be effective for polystyrene used in microplate ELISA assays. Because of physical binding properties, these reagents are insufficient for blocking nylon and PVDF membranes. They can, however, still be used with nitrocellulose. The best blocking agent for all membranes has been found to be milk casein, commonly used in buffers as either 2% dried milk or 0.5% Hammersten grade casein. These agents will work with proteins and nucleic acids and will usually provide excellent backgrounds with all detection systems.

In some cases, background signal persists despite blocking with casein. The amount of background generated is usually tied to the detection reagents used. Background can be decreased by a variety of means, including decreasing probe and conjugate concentration, changing substrates or changing membrane type. This catalog has several articles devoted to optimizing procedures and membrane choice in order to obtain the best balance between sensitivity and background.

Pall Corporation manufactures membranes made from nitrocellulose, nylon, and PVDF for molecular detection applications. Nylon membranes are available with neutral, positive, and negative surface charge. Activated surfaces designed for covalent attachment of proteins and nucleic acids are also available. Learn more about Pall's detection membranes.