The Western 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
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
of Western blot background and solutions.
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
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
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
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
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
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
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.
i. Start dirt-free
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.
b. Air Bubbles
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
i. 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.
ii. 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
c. Poor Handling of Membrane
Splotches, lines and dark spots often arise due to poor handling of membrane.
i. Membrane manipulation
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.
ii. 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
d. 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.
e. 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.
f. Excessive Detection Reagents on Blot
If detection reagents pool unevenly on the membrane the can cause a splotchy
Bands for troubleshooting Western blots with multiple discrete bands. Also, see our
blog for a quick Western