Membranes used for Western blotting have a high affinity for proteins allowing binding and retention of transferred proteins. Before transferred proteins can be detected using antibodies, the membrane must be incubated with a blocking buffer to block the unoccupied binding surfaces on the membrane. Efficient blocking will allow antibodies to access target proteins bound to the membrane while preventing high background due to nonspecific binding of primary and secondary antibodies.
The most important consideration when choosing a blocking solution is the signal:noise ratio. Optimizing blocking conditions can enhance the specific signal for an antigen-antibody interaction while decreasing background noise significantly. Efficient blocking increases the signal:noise ratio leading to a strong specific signal while reducing background staining. Inadequate blocking results in excessive background and lowers the signal:noise ratio. Excessive blocking can inhibit antibody:antigen interactions or in interfere with detection reagents, also lowering the signal:noise ratio.
Although blocking the membrane is one of the most crucial steps in Western blotting, the choice of which blocking agent to use is often overlooked as a step in optimizing Western blots. Not all blocking agents are compatible with every antigen-antibody combination and adequate blocking depends on the antigen and the type of detection agent used downstream.
Below is a guide to different blocking agents.
Blocking buffers are composed of a salt solution, with or without detergent, and a blocking agent. Salt solutions include Tris-buffered saline (TBS; 50 mM Tris and 150 mM NaCl, pH 7.6) and phosphate-buffered saline (PBS; 140 mM NaCl, 10 mM phosphate buffer, and 3 mM KCl, pH 7.4). While both of the salt solutions can usually be used interchangeably, specific conditions may require the use of one buffer instead of the other. For example, PBS can interfere with alkaline phosphatase (AP) so TBS should be used when AP is used as a substrate downstream. A mild detergent (Tween 20, 0.05%) can also be added to the solution as a blocking agent.
Note: Detergent-containing solutions can promote microbial growth and blocking buffer should be made fresh prior to use.
Blocking agents are molecules used to saturate free binding sites on the membrane, preventing nonspecific binding of primary and secondary antibodies in downstream steps. Blocking agents work by covering the unoccupied areas of the membrane with a dense layer of molecules. Blocking agents can either contain proteins, or be protein-free.
Protein-containing blocking agents
Some of the most popular blocking agents are those that contain proteins. As membranes have a high binding capacity for protein, these blocking agents readily adsorb to all unoccupied sites on the membrane. While generally inexpensive and readily available, protein blocking agents can interfere with some antibody-antigen interactions. Additionally, some proteins may be directly recognized by primary or secondary antibodies causing high background. This issue can be overcome by using another type of protein blocking agent or by using a protein-free agent. Some of the more common protein blocking agents are described below.
Non-fat milk is one of the most common blocking agents used for Western blots. It contains a variety of proteins found in milk. Non-fat milk is relatively inexpensive and is easily prepared in the lab from stocks of dry powder. Milk solutions should be filtered prior to use to prevent particulates from binding to membrane and causing a “speckled” background.
Overall, non-fat milk is a good first choice for a blocking agent. However, milk should never be used when detecting a phosphorylated protein. Milk contains casein, a phosphoprotein that can be recognized by anti-phospho antibodies leading to non-specific binding and high background. Milk also cannot be used if avidin-biotin detection systems are used as milk contains biotin.
Most protocols suggest starting with 5% non-fat milk. However, high concentrations of milk can mask some antigens, particularly if they are in low abundance. It is more advantageous to begin with lower concentrations of milk such as 1%.
Bovine serum albumin (BSA)
BSA (sometimes called Fraction V) is another commonly used protein blocker derived from the serum of cows. Similar to milk, it is a good general blocking agent that is easily prepared in the lab. Similar to milk-containing buffers, BSA solutions should be filtered to remove particulates.
As mentioned above, BSA is usually preferred over milk when detecting phosphorylated proteins. However, some BSA preparations contain tyrosine phosphorylations and will give a high background when using anti-phosphotyrosine antibodies. Moreover, BSA is not compatible with lectin probes as it contains carbohydrates that can increase non-specific background.
BSA can be used in a range from 0.3-5% depending on the application.
Serum (horse or fetal calf)
Whole serum, which contains multiple proteins, can be used as a blocking agent at a 10% concentration. The serum is usually derived from horse or fetal calf. Serum is more expensive than milk or BSA and is less commonly used as a blocking agent.
Whole serum contains immunoglobulins that can potentially cross-react with primary or secondary antibodies leading to high, non-specific background.
While more commonly used in immunohistochemistry, 2% fish gelatin can be used as a blocking agent for Western blots. Gelatin is more expensive than BSA and milk, but does not cross react with mammalian proteins.
Gelatin cannot be used with avidin-biotin detection systems due to endogenous biotin. While it can decrease the background in some applications, fish gelatin can also mask some antigens.
Single purified protein
Purified single proteins can be used as blocking agents and many are available commercially. The use of a single protein as a blocking agent can prevent cross-reactivity with other components seen when milk or whole serum is used. Different proteins can be empirically tested for each type of Western blot. Whole casein protein (1%) isolated from milk is an example of a single protein used as a blocking agent. Casein protein cannot be used when detecting phosphoproteins (as mentioned above).
Protein-free blocking agents
While protein-containing blocking agents are widely used in Western blots, they can have some disadvantages. As discussed above, some proteins, such as casein, can react with antibodies resulting in high background. Other blocking agents can interfere with detection substrates or mask target proteins. For this reason, several companies have developed protein-free blocking agents to provide efficient blocking without cross reactivity or interference. For example, Advansta sells AdvanBlock-PF.
AdvanBlock-PF is a proprietary protein-free blocking solution compatible with both chemiluminescent Western blots and fluorescent Western blots. AdvanBlock-PF can reduce background when used with antibodies that have a high degree of cross-reactivity with protein blocking agents. AdvanBlock-PF can also be used with protein blocking agents to increase blocking efficiency for low quality primary antibodies.