Good Western blot blocking methods are crucial in order to achieve clean and reliable results. Protein samples are first ran on a polyacrylamide gel to separate proteins by size. The proteins are then transferred to a nitrocellulose or PVDF membrane. A blocking agent is then used to prevent primary and secondary antibodies from binding to the membrane non-specifically (areas without the protein of interest). Proper blocking methods cover the membrane surface without interfering with bound protein; thus preventing non-specific antibody binding and non-specific signal (noise or background) due to the membrane’s high affinity for proteins.
There are three factors to consider for correct blocking:
- Blocking method
- Blocking agent
- Buffer
Below are some tips and tricks to keep in mind to ensure beautiful western blot results.
Use Proper Blocking Methods
- Shake the membrane in chosen blocking agent at an appropriate speed (not too fast/not too slow) for 1 hour at room temperature. Shaking is necessary for many western blot steps (blocking, washing, primary/secondary antibody incubation). The correct speed will ensure uniform blocking of unoccupied binding sites on the membrane which prevents non-specific binding of primary/secondary antibodies.
- Make sure that the membrane is placed “protein side up” with blocking buffer gently washing over it.
- Fully dissolve blocking buffer: Blocking buffers are often prepared in a hurry, but improperly dissolved buffers can cause black dots on your western blot. Prepare blocking buffer ahead of time to avoid this. If black dots persist, then filter your blocking buffer before use to remove particulates.
Choice of Blocking Agent
There are three factors to consider when choosing an appropriate blocking agent: antibody compatibility, compatibility of the protein of interest, and the detection system. Some common blocking buffers and tips for their use are listed below.
- Non-fat Milk powder (2.5-5% solution) is the most commonly used and cheapest blocking agent. Milk should not be used if the protein of interest is phosphorylated. Milk contains casein, a phosphoprotein that will bind to anti-phospho antibodies which causes non-specific binding and high background noise. Milk may mask some antigens if they are in low abundance which would cause faint bands. If faint bands occur then decrease concentration to 1% in blocking and antibody solutions or substitute with 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. However, BSA preparations contain tyrosine phosphorylations and will cause high background noise with anti-phosphotyrosine antibodies. Filter BSA before use. Unfiltered BSA may contain contaminating IgG or serum proteins which can cause background noise. BSA is not compatible with lectin probes because it contains carbohydrates that will increase non-specific binding.
- Fish Gelatin is more commonly used for immunohistochemistry, but can be used for Western blots at 0.1-5% concentrations. Fish gelatin is purified from cold-water fish and can remain liquid at colder temperatures. Fish gelatin does not cross-react with mammalian antibodies because it does not contain mammalian serum proteins. Although, do not use fish gelatin with avidin-biotin detection systems because it contains endogenous biotin.
- Normal Serum (bovine, rabbit, goat, mouse, etc) is less common because it is used at higher concentrations (5-10%) and is more expensive than milk or BSA. Normal serum carries immunoglobulins that bind to reactive sites on the membrane which prevents nonspecific binding of the labeled IgG antibodies. Use serum from the same species as the secondary antibody. Be sure not to use serum from the same species as the primary antibody because this would cause non-specific binding of the primary antibody across the membrane.
- Proprietary Commercial Buffers contain pure fractions of proprietary protein without albumin, immunoglobulins, phosphoproteins or biotin. Commercial buffers work efficiently and yield high quality results so less time is spent blocking. Specific protocols for each commercial buffer are available.
- Protein Free Blocking Agents
- Polyvinylpyrrolidone (PVP) is a non-protein blocking buffer alternative that is useful for detecting small proteins. PVP is a water-soluble polymer that binds to nitrocellulose and PVDF membranes. PVP is generally used at 0.5-2% concentration and is commonly combined with purified casein or other blocking agents.
What Buffer to Use?
Blocking buffers are used to dilute the blocking agent and antibodies. They are composed of a salt solution, with or without detergent (Tween 20, 0.05%), and the 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). For most applications they can be used interchangeably, but specific conditions may require the use of one buffer over of the other.
- PBS can interfere with alkaline phosphatase so TBS should be used if alkaline phosphatase is used as a downstream substrate.
- Low concentrations of Tween 20 in the blocking buffer and antibody buffer is used to prevent weak non-specific binding. The specific binding of antibodies is strong and should not be affected.
- Detergent-containing solutions can promote microbial growth so TBS or PBS with Tween should be prepared fresh prior to use.
- For optimal assay conditions, use the same blocking buffer for diluting the antibody that is used for the blocking step.
For other tips and tricks for better Western blotting review: