In Western Blot, selecting the proper membrane is critical to the success of protein transfer procedure. Membranes may differ according to the type and manufacturer and users generally have to rely on advertised claims and generalisations about membrane properties to decide what is the most suitable membrane for an application. Experience shows that the performance of membranes in assays involves other factors, such as the nature of the protein which is to be bound, the buffer used whether the proteins have been reduced, the capacity of the membrane to retain the protein, non-specific binding of other proteins and ligands to the membrane (which may create an unwanted background) and the rate at which the membrane will bind the proteins presented to it (unpublished observations).
Considering all of this, the following contrasts the features of three types of membranes, i.e., PVDF, nitrocellulose and nylon, hoping it will give you a general view of what these membranes are capable of and help you choose the right membrane for your Western blotting needs.
|Protein size||Better for high MW proteins||Better for mid to low MW proteins (< 20 kDa)||Better for nucleic acids|
|Binding capacity||100 - 200 µg/cm2||80 - 100 µg/cm2||> 400 µg/cm2|
|Background||Slightly low||Very low||High|
|Strip & Re-probe||Performs well||Possible, but can lose sensitivity||Performs well|
|Mechanical strength||High||Brittle when dry||High|
|Pre-treatment||Wet by methanol||Wet by buffer||Wet by buffer|
|Pore size||0.2 μm, 0.45 μm||0.2 μm, 0.45 μm||0.45 μm|
|Applications*||WB, SB, NB, PS, AA||WB, SB, NB, AA||WB, SB, NB|
PVDF (Polyvinylidene fluoride) membranes are mechanically strong. The mechanism of protein binding to the membrane is by hydrophobic interaction. Wetting the PVDF membrane with methanol prior to transfer shown to have increase the protein binding. Proteins binds more tightly to the PVDF membrane than to the Nitrocellulose membranes. The tight binding helps in retaining the protein during the washing steps. PVDF is considered to be one of the low protein binding membranes.
Nitrocellulose membranes, which were introduced for use in protein blotting in 1967, are one of the most commonly used blotting membranes now. The mechanism of nitrocellulose membranes binding to proteins is probably due to hydrophobic interactions, high salt and low methanol concentrations, improving protein immobilization to the membrane during electrophoretic transfer. The main advantage of using nitrocellulose membrane is that which gives very low background. However, nitrocellulose membranes have low mechanical strength, and become brittle and difficult to handle when dry. This limits their use for multiple stripping and reprobing in western blot experiments.
Nylon (polyamide) membranes are made up of unmodified or positively charged Biodyne A and Biodyne B nylon. The mechanism of this membranes binding proteins and nucleic acids are ionic, electrostatic and hydrophobic interactions. Nylon membranes have high mechanical strength, which offers benefits in western blot experiments requiring stripping and reprobing procedures. Nylon membranes consistently provide a higher efficiency of protein transfer SDS gels than nitrocellulose membranes. However, a significant drawback to using nylon membranes for blotting applications is that they bind the common anionic dyes such as Coomassie blue and Amido black which may result in background staining that can obscure the detection of transferred proteins.