Western blotting is a laboratory technique used to detect specific proteins within a sample. This process involves separating proteins by size, transferring them to a solid support, and then using antibodies to identify the target protein. The membrane, which captures proteins after separation, is a central component of this technique. Proper storage of these membranes is important for preserving experimental results, allowing for re-analysis, and maintaining protein signal integrity over time.
Preparing Membranes for Storage
Proper preparation immediately following Western blot is essential for successful membrane storage. Thorough washing removes excess reagents (e.g., antibodies, detection substrates) that can degrade or interfere with future analysis, preventing issues like increased background noise or protein degradation.
For many storage methods, especially long-term ones, thoroughly drying the membrane is important to prevent microbial growth and protein degradation. Different membrane types, such as polyvinylidene difluoride (PVDF) and nitrocellulose (NC), have distinct properties that influence this initial preparation. PVDF membranes are more robust and can be dried completely, but they require re-wetting with methanol before subsequent use. Nitrocellulose membranes, on the other hand, can become brittle if fully dried and are often stored wet or only partially dried to maintain their integrity.
Short-Term Storage Approaches
For short-term storage (a few days to two weeks), refrigeration is a common and effective method. Keeping membranes at 4°C slows down enzymatic activity and microbial growth, which helps preserve protein integrity and signal. Membranes can be stored wet in buffers like phosphate-buffered saline (PBS) or Tris-buffered saline (TBS).
Wet storage in buffer maintains hydration, preventing the membrane from drying out and becoming brittle, especially for nitrocellulose. Some protocols suggest adding a preservative like sodium azide to prevent bacterial contamination, though it must be thoroughly washed out before re-probing as it can inhibit horseradish peroxidase (HRP) activity. Alternatively, membranes can be stored dry between protective sheets, which also helps prevent degradation over the short term.
Long-Term Preservation Techniques
For extended preservation, often spanning months or even years, freezing membranes is a widely used technique. Storing membranes at temperatures such as -20°C or -80°C significantly inhibits enzyme activity and microbial proliferation, offering more robust protection against degradation compared to refrigeration. PVDF membranes are preferred for long-term storage due to their chemical resistance and stability.
When freezing, membranes can be stored either wet or dry. Dry storage, typically by air-drying and sealing in a protective layer or vacuum bag, is often recommended for PVDF membranes to prevent ice crystals that could damage protein or membrane structure. For nitrocellulose, which can become brittle when fully dried, some prefer storing them moist or with minimal drying before freezing. Regardless of wet or dry, membranes should be placed between protective sheets and sealed in plastic bags to prevent desiccation, contamination, and light exposure.
Addressing Storage Challenges
Improper storage can lead to several issues, including signal loss, increased background noise, and protein degradation, all of which compromise the reliability of future analysis. Signal loss can occur if proteins detach from the membrane or degrade over time, often exacerbated by incomplete washing of reagents that might continue to react or break down. Increased background noise can result from residual antibodies or detection substrates not fully removed prior to storage, leading to non-specific binding upon re-probing.
Protein degradation can also stem from incomplete drying, which allows residual enzyme activity or microbial growth, or from temperature fluctuations during storage. Repeated thawing and freezing cycles are particularly damaging, as they can cause physical stress to the membrane and proteins, leading to protein loss or aggregation. To avoid these issues, ensuring membranes are thoroughly washed and dried (if applicable), maintaining consistent storage temperatures, and minimizing freeze-thaw cycles are important practices for preserving the membrane’s viability for future use.