A Western blot is a widely used laboratory technique that allows scientists to identify and quantify specific proteins within a complex mixture of biological samples. A crucial step in this process is blocking.
The Purpose of Blocking in Western Blot
Following the transfer of proteins from a gel to a membrane, the membrane surfaces become highly receptive to protein binding. This means that not only the target protein but also the antibodies used for detection could non-specifically attach to the membrane.
To counteract this, blocking is employed to saturate all unoccupied binding sites on the membrane. This saturation ensures that when antibodies are introduced, they bind almost exclusively to the specific target protein. This saturation process effectively minimizes background noise. The blocking step enhances the clarity and specificity of the detection. A properly blocked membrane leads to a cleaner signal.
Key Components for Blocking Buffer
Blocking buffers typically consist of a protein-based blocking agent dissolved in a buffer solution. The two most common protein-based blocking agents are non-fat dry milk and Bovine Serum Albumin (BSA). Non-fat dry milk is a widely used and economical option, readily available and simple to prepare. However, it contains intrinsic biotin and phosphatases, which can interfere with the detection of phosphorylated proteins or when using biotin-conjugated antibodies, potentially leading to high background or false positives.
Bovine Serum Albumin (BSA) is another popular blocking agent, known for providing clearer results due to its higher purity and containing fewer cross-reactive proteins. BSA is particularly recommended when detecting phosphorylated proteins, as it lacks the phosphatases found in milk that can dephosphorylate target proteins. While generally more expensive than non-fat dry milk, BSA is a versatile blocking agent suitable for many applications.
These blocking agents are typically dissolved in a buffered saline solution, such as Phosphate-Buffered Saline (PBS) or Tris-Buffered Saline (TBS). These diluents are crucial as they maintain the appropriate pH and ionic strength, creating a stable environment for the blocking agents and subsequent antibody incubations. Sometimes, a small amount of a non-ionic detergent like Tween-20 is added to the buffer, which further aids in reducing non-specific binding by disrupting weak interactions.
Step-by-Step Blocking Buffer Preparation
Preparing blocking buffer is a straightforward process, typically involving dissolving the chosen blocking agent in a buffered saline solution. A common concentration for both non-fat dry milk and BSA is 5% (weight/volume). For example, to prepare 100 milliliters of 5% blocking buffer, you would measure out 5 grams of non-fat dry milk powder or BSA. This amount is then added to 100 milliliters of the chosen buffer, such as 1X TBS or 1X PBS.
After adding the powder to the buffer, the mixture must be thoroughly stirred or agitated to ensure complete dissolution. This usually takes at least 30 minutes on a magnetic stirrer to prevent clumping. It is also advisable to use purified water for preparing the buffers to avoid contaminants. Once dissolved, the solution can be filtered to remove any undissolved particulates, which helps prevent speckles or dark spots on the membrane during subsequent detection steps. If desired, 0.1% Tween-20 can be incorporated into the buffer during preparation to enhance its blocking efficiency.
Optimizing Your Blocking Strategy
The choice of blocking agent, its concentration, and incubation conditions all play a role in the effectiveness of the blocking step. For instance, while non-fat dry milk is a versatile and cost-effective option for many applications, BSA is generally preferred when working with phospho-specific antibodies or biotin-conjugated detection systems due to milk’s inherent components. Always consult the antibody datasheet for recommended blocking agents, as some antibodies may be sensitive to certain components.
The optimal concentration of the blocking agent typically ranges from 3-5%, though adjustments may be necessary depending on the specific experiment. Incubation time is another factor that can be optimized; membranes are commonly blocked for 30 minutes to 1 hour at room temperature with gentle agitation. For more sensitive proteins or to ensure thorough blocking, an overnight incubation at 4°C can also be effective.
Proper storage of prepared blocking buffer is also important. While freshly prepared buffer is always recommended, a BSA-based blocking buffer can typically be stored at 4°C for up to a week. Milk-based blocking buffers are generally less stable and are best prepared fresh on the day of the experiment or used within a few days if stored cold. High background or weak signals on a blot can often be traced back to issues with the blocking step, such as incomplete blocking or interference from the blocking agent itself.