NC Membrane, 0.22 μm
Nitrocellulose (NC) transfer membrane is widely used for protein and nucleic acid detection in research and medical diagnostics, and is one of the classic blotting supports. NC membrane offers advantages such as relatively high protein binding capacity, no pre-activation requirement, and low background, making it a common choice for routine Western blot applications. It is suitable for various applications including Western blotting, Northern blotting, Southern blotting, and dot/slot blotting.
NC membrane is inherently hydrophilic and can be directly wetted in transfer buffer without the need for pre-wetting with organic solvents such as methanol, offering ease of operation and relatively improved safety. Its protein binding capacity is typically approximately 0.08–0.11 mg/cm² (80–110 μg/cm², exact values vary slightly by brand and model), with binding mechanisms primarily involving hydrophobic interactions and electrostatic forces, and minimal further perturbation to protein spatial conformation. NC membrane generally exhibits clean background and high signal-to-noise ratio, making it suitable for routine single-use Western blot detection, as well as various colorimetric, chemiluminescence, or fluorescence detection systems. The disadvantages of NC membrane include becoming brittle after drying, lower mechanical strength and chemical stability compared to PVDF membrane, poor tolerance to many organic solvents, and unsuitability for frequent or vigorous stripping and multiple rounds of reprobing.
NC membranes are commonly available in 0.22 μm and 0.45 μm pore sizes: the 0.22 μm pore size is more suitable for small molecular weight proteins (typically < 20 kDa), while the 0.45 μm pore size is recommended for most routine proteins (> 20 kDa). Compared with PVDF membranes, NC membranes are more economical and represent a cost-effective choice that balances cost and operational convenience.
Low background, high signal-to-noise ratio, and excellent detection sensitivity
Hydrophilic material – no methanol pre-activation required
Compatible with a variety of detection methods, including colorimetric, chemiluminescence, and fluorescence
High protein binding capacity, suitable for routine single-use Western blotting
Cost-effective with relatively low price
Lower mechanical strength and organic solvent resistance compared to PVDF; not suitable for multiple stripping and reprobing
Q: What are the advantages and disadvantages of NC membrane compared with PVDF membrane?
A: Both membranes are commonly used solid supports in protein blotting. NC membrane offers low background and high signal-to-noise ratio, but is fragile and less durable. PVDF membrane is more durable with stronger protein binding capacity, but requires an additional activation step.
Q: How do I choose NC membrane pore size based on protein molecular weight?
A: NC membranes are commonly available in 0.22 μm and 0.45 μm pore sizes. The 0.22 μm pore size is more suitable for small molecular weight proteins (typically < 20 kDa), while the 0.45 μm pore size is recommended for most routine proteins (> 20 kDa).
Q: Does NC membrane require methanol activation before use?
A: No. NC membrane is hydrophilic and can be naturally wetted by aqueous buffers without the need for soaking in ethanol, methanol, or other organic solvents. In contrast, PVDF membrane is highly hydrophobic and must be pre-activated with methanol or ethanol for effective protein binding. If the NC membrane does not wet completely in water or transfer buffer, it can be soaked in 0.1% Triton X-100 for approximately 5–30 minutes without affecting subsequent experiments.
Q: Which detection methods are compatible with NC membrane?
A: NC membrane offers broad compatibility and can be used with chemiluminescence (e.g., ECL), colorimetric detection (e.g., TMB), staining (e.g., Ponceau S staining), and isotopic detection, as well as fluorescence detection. However, note that NC membrane has higher autofluorescence than low-fluorescence PVDF membranes, which may affect sensitivity and accuracy in fluorescence-based detection.
Q: Can NC membrane be used for nucleic acid detection?
A: Yes. NC membrane can also be used for nucleic acid blotting (e.g., Northern blot, Southern blot), with a binding capacity of approximately 80–100 μg/cm² for DNA and RNA. However, its binding efficiency for short nucleic acid fragments (e.g., < 200 bp) is relatively low. Nylon membranes are more commonly used for nucleic acid detection due to their higher binding capacity (480–600 μg/cm²) and ability to retain fragments as short as 10 bp.