Table of Contents
Is milk or BSA better for blocking?
Many researchers choose to use Milk over BSA during as their blocking agent as it is the cheaper and easier option available. Its general application is for experiments where the antibodies have a good binding ability and the target protein is expressed at relatively high levels.
Why is a blocking solution such as BSA or milk applied to the membrane after transfer?
Blocking is a very important step of western blotting, as it prevents antibodies from binding to the membrane nonspecifically. Blocking is often made with 5\% BSA or nonfat dried milk diluted in TBST to reduce the background. Nonfat dried milk is often preferred as it is inexpensive and widely available.
What is the function of the blocking agents non fat milk?
Types of blocking buffers
| Blocking Buffer/Agent | Benefits |
|---|---|
| Purified proteins (e.g., casein) | Single-protein blocking buffers can provide fewer chances of cross-reaction with assay components than serum or milk solutions. Ideal when blockers, such as non-fat milk, block antigen-antibody binding |
What is BSA blocking buffer?
Bovine serum albumin (BSA) blocking buffer is ideal for saturating excess protein-binding sites on membranes and microplates for Western blotting and ELISA applications, respectively. Typically, 1-3\% BSA is sufficient for most applications.
Why is milk used for blocking?
Overall, non-fat milk is a good first choice for a blocking agent. 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.
Why is BSA used as blocking agent?
How Does BSA Work as a Blocking Agent? Blocking agents are meant to prevent the nonspecific binding of your antibodies. In general, as long as a protein doesn’t have affinity for your probe, it can theoretically be used as a blocking agent.
Should you filter BSA?
Bovine Serum Albumin (BSA): Purified albumin from bovine serum is the second most common blocking agent and is used in a 2-5\% concentration. BSA can be used to detect phosphorylated proteins. Filter BSA before use. Unfiltered BSA may contain contaminating IgG or serum proteins which can cause background noise.
How does BSA blocking work?
BSA blocking is a routine practice among clinicians and researchers working on immunoassays throughout the world. The primary role of BSA is to prevent the non-specific binding by blocking the leftover spaces over solid surface after immobilization of a capture biomolecule.
How does BSA block nonspecific binding?
Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein – typically 3-5\% Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in Tris-Buffered Saline (TBS) or I-Block, with a minute percentage (0.1\%) of detergent such as Tween 20 or Triton X-100.
What does milk protein in blocking buffer do?
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.
How does BSA block non specific binding?
How does milk block the membrane?
Non-fat milk is one of the most common blocking agents used for Western blots. 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.
What is the purpose of using non-fat milk?
Actually, the purpose of using non fat milk is to block the high affinity areas on membrane.
Can I use BSA instead of milk as a protein blocker?
However, if you are using phospho-antibodies, start with BSA and if that doesn’t work switch to milk. If all else fails, you could try a non-protein blocking agent such as WesternBreeze Blocker, Pierce Protein-Free, or many more which are commercially available.
What is the difference between BSA and single-protein blocking buffers?
BSA is generally a weaker blocker, which can result in more non-specific antibody binding, but can increase the detection sensitivity for low-abundant proteins. Single-protein blocking buffers can provide fewer chances of cross-reaction with assay components than serum or milk solutions.
Should primary antibodies be diluted in 5\%BSA or 5\% non-fat milk?
Some protocols mention primary antibodies should be diluted in 5\%BSA whereas some protocols mention primary antibodies should be diluted in 5\% non-fat milk. What is the difference?