The Westerns blot assay is a powerful technique to analyze protein expression. With this single assay, individual proteins can be assessed for molecular weight, post-translational modifications and abundance. Western blots are relatively simple to perform and do not require expensive equipment or reagents, making them the mainstay for many labs.
The key to a successful Western blot is to use an antibody that specifically reacts with a single protein and has little cross-reactivity with other proteins. A successful blot also relies on obtaining a signal:noise ratio that allows detection of the protein with minimal background. High uniform background and splotchy uneven background are common problems in Western blots. This wiki entry describes common causes and solutions for high background in Western blots.
Uniform High Background
Uniform high background is background that darkens the entire blot making it difficult to see specific bands. Uniform background can result from a variety of reasons.
High Concentration of Antibodies
If excessively high concentrations of antibodies are used, they can saturate and coat the membrane, binding non-specifically. This will lead to a uniform high background over the entire blot. When using enhanced chemiluminescence (ECL), sometimes the blot will even glow in the dark.
To reduce high background due to high antibody concentration, titrate the antibodies. A dot blot can be used to titrate both primary and secondary antibodies in a checkerboard like pattern.
Blocking the membrane is a crucial step that prevents non-specific binding of antibody to the membrane. Sufficient blocking is achieved by incubating the blot in an appropriate blocking buffer. Type of blocking agent, length and temperature of incubation can affect efficiency of blocking.
The choice of blocking reagent depends upon the particular antigen:antibody interaction and is best determined empirically.
Non-fat dry milk is a commonly used as the first choice. It is often recommended that 5% milk be used for blocking, however sometimes this concentration of milk can mask the protein being detected, particularly if it is not abundant. We recommend starting with 1% milk.
Milk can be incompatible with certain antibodies and detection systems (see Interference from incompatible blocking agents), and in this case, 3% bovine serum albumin (BSA) is a second protein-based blocking agent that can be used.
Several companies market protein-free blocking agents such as AdvanBlock-PFby Advansta. Protein-free agents prevent interference, but also can be used in combination with protein-based agents to increase blocking capacity.
Length and temperature of incubation
Blocking for 1 hour at room temperature with agitation is usually sufficient to block the membrane. However, blocking can also be performed overnight at 4°C with agitation.
Interference from incompatible blocking agents
Uniform high background can be observed when blocking agents interact with antibodies causing interference. For example, antibodies can bind to proteins in protein-based blocking agents and result in high uniform background. In particular, animal serum containing unpurified primary antibodies can bind to animal proteins in blocking reagents. In addition, non-fat dry milk can specifically react with certain antibodies or detection reagents. Milk contains casein, a phosphoprotein that can react with phospho-specific antibodies. Milk also contains variable amounts of avidin that can interfere with avidin-biotin detection systems. Both of these interactions will result in high uniform background.
Test different blocking reagents
The appropriate blocking agent should be determined for each antigen:antibody combination. As discussed above, the two most common blocking agents are non-fat dry milk and bovine serum albumin. If neither of these agents is adequate, then protein-free or non-animal protein blocking reagents, such as AdvanBlock can be used. Alternatively, normal serum that is from the same species as the animal that the primary antibody was produced in can be used as a blocking reagent.
It is important to wash the blot sufficiently after incubation with primary and secondary antibodies to remove excess antibodies. Poor washing can contribute to high uniform background.
Change volume, length and number of washes
Perform washes in a large volume, change the wash solution often and agitate the membrane during the washing steps. A recommended protocol is to wash the membrane 3 times after each incubation, washing for 5 minutes per wash. The number of washes and length of wash time can be increased to improve washing efficiency; however overwashing can decrease signal strength.
Increase stringency of washing with detergent
The stringency of the washing step can be increased by including detergent in the wash solution to disrupt non-specific interactions. Tween-20 (0.05%) is commonly used in wash buffers. A slightly stronger detergent, such as 0.05% NP-40 can be used to increase stringency of the wash. Note: Only highly purified detergents should be used as impurities in detergents can interfere with horse radish peroxidase detection systems. Detergent should be freshly added to wash solutions as detergents can promote the growth of bacteria and cause high background (see Bacterial Contamination).
Different membrane types (nitrocellulose versus PVDF) and vendor and/or lot sources of membranes can affect the level of background. The age of the membrane and how membranes are handled can also impact the degree of background obtained.
The choice of membrane should be determined empirically for each antigen:antibody combination. Nitrocellulose membranes tend to give the lowest background, however they are brittle, cannot be stripped and reprobed and many not bind smaller proteins. Polyvinylidene difluoride (PVDF) membranes have a high binding capacity, but may give higher background.
PVDF membranes require activation in methanol and equilabration in water prior to use. High uniform background will be seen if these steps are omitted or not performed appropriately. Refer to the manufacturer’s instructions for a protocol.
Membranes should never be allowed to dry out as this will also result in high uniform background.
The use of precut membanes, such as the precut PVDF or nitrocellulose membranes sold by Advansta can minimize handling and contact with dirty scissors.
Source of membrane
Background levels can also vary depending upon the vendor source of membranes. If high background is seen, a different source can be tried to lower background.
Age of membrane
Older membranes can give high uniform background, therefore pay attention to expiration dates and use newer membranes to decrease background.
Bacterial contamination of buffers can lead to high background.
Buffers containing detergent and milk, which can promote bacterial growth, should be made fresh.
Overexposure of membrane
Long exposure times will enhance overall background, without increasing sensitivity.
Several options can be tried to decrease exposure times. If possible, load more protein to increase abundance of the target antigen. Make sure the primary antibody is titrated well. Try different primary antibodies if available. Alternatively, use a detection reagent designed for low abundance proteins such as Advansta’s WesternBright Quantum HRP substrate.
Blotchy, Uneven or Speckled Background
In addition to having high uniform background, Western blot background can include uneven splotchy background, random black smudges, white spots or cleared areas, uneven bands, and random speckles. There are several reasons for this type of background.
Chemiluminescent reagents are attracted to dust and dirt that may fall onto the blot during washes or be transferred to the blot from dirty equipment. Background from dust and dirt is usually seen as random speckles on the blot.
To prevent dust and dirt contamination, wash all equipment that comes in contact with the membrane, this includes the transfer apparatus and trays used for incubations. Filter all buffers and solutions to remove particles. Keep covers on dishes to prevent dust from falling into solutions during incubations.
If air bubbles are on the membrane during transfer, they will prevent proteins from adhering to the membrane. If bubbles attach to the membrane at a later time, they can prevent the blocking solution or antibodies from accessing the membrane. Air bubbles can cause splotchy blots, white circles or cleared areas, and incomplete bands.
Air bubbles during transfer set-up
To prevent air bubbles when setting up the transfer, gently roll a pipette over each portion in the “sandwich” stack (membrane, filter paper, transfer sponge) after it has been put in place.
Air bubbles during transfer and incubations
Air is created in the transfer buffer due to the mixing of methanol with aqueous buffer. When possible, prepare transfer buffer 1-2 days in advance. If fresh buffer has to be used, degas the buffer by filtration, vacuum or sonication. In addition, transfer at a lower voltage to prevent bubbles.
Avoid agitating the blot too vigorously during incubations and creating bubbles.
Poor Handling of Membrane
Splotches, lines and dark spots often arise due to poor handling of membrane.
Always handle membranes with gloved hands and use forceps for manipulations. Oils on the hand will cause dark splotches. Be careful not to fold the membrane making a crease, this will cause dark lines. Do not scratch the membrane when adding solutions or using forceps. Make sure the membrane is saturated with solutions during incubations to prevent areas from drying out.
Activation and equilibration of PVDF
When using PVDF, it is important to activate the membrane and then equilibrate the membrane in an aqueous solution, as described above. When methanol reacts with aqueous solutions, air bubbles are created that may stick to the membrane and prevent equilibration and diffusion, giving a splotchy background. Make sure the membrane is equilibrated following the manufacturer’s instructions. If there is a temperature difference between the solutions (i.e. water is cold and methanol is at room temperature), then the methanol might not be displaced adequately; therefore try to maintain even temperature when preparing the membrane.
Insufficient Mixing of Buffer Components
Buffer components, such as detergents and blocking agents can stick to the membrane and cause dark spots if they are not completely solubilized. This is particularly common with non-fat dry milk.
Completely mix buffer components
Make sure all solutions are mixed thoroughly before use; blocking solutions can be vortexed. Solutions can also be filtered before use.
Antibody not Evenly Distributed During Incubation
Uneven splotchy background or incomplete bands can occur if the antibody is not evenly distributed over the membrane during incubations.
Keep membrane covered with solutions
Use enough antibody solution to completely cover the membrane. Agitate the blot on a shaker during incubations.
Excessive Detection Reagents on Blot
If detection reagents pool unevenly on the membrane the can cause a splotchy background.
Remove excess detection reagents
To remove excess detection reagents carefully blot the protein side of the membrane with a Kim-wipe or drop the membrane onto filter paper quickly to wick off liquid. Wrap the membrane in plastic wrap prior to exposure to prevent further drying.
Photo courtesy of Agnes Periapse.