Western Blot Troubleshooting Tips


Western Blot result depends on the whole system including antigen content, sensitivity of primary antibody, sensitivity of secondary antibody, sensitivity of substrate, efficiency of color development and photographic fixing. Mistake of any procedure in the experiment may lead to unsatisfactory results.

Here Elabscience lists the common Western Blot troubleshooting.

1. No band or low band

Possible causes


The loading amount of sample is low

Increase the loading amount

The target protein quantity is too low or not expressed in the sample

Refer to the relevant literatures to make sure that the sample contain your target protein, or prepare fresh sample again and choose a positive sample as control

There is a poor transfer of protein to membrane or not enough protein is bound to the membrane

Ensure that the transfer order is correct and the transfer time is sufficient

The antibody concentration may be too low

Use a higher concentration of antibody

The antibody concentration may be too high to cause the signal to disappear instantly

Use a lower concentration of antibody

Insufficient exposure time

Prolong the exposure time

Insufficient incubation or deactivation of the substrate

Increase the incubation time of substrate and ensure that the substrate is valid

The target protein transferred to the membrane has degraded

Keep a low transfer temperature, decrease the transfer electric current and transfer time

Excessive washing of the membrane

Reduce the frequency or duration of washing steps

The antibody is inactive or the titer of antibody is too low

Pay attention to the preservation of antibody and use antibody with higher titer

2. High background

Possible causes


The experimental equipment has been contaminated

Ensure that the equipment is clean

Some membrane may cause high background

NC membranes are considered to cause less background than PVDF membranes.

Blocking buffer is not compatible or there is cross-reactivity between the blocking buffer with antibodies

Replace the blocking buffer

Insufficient blocking

Prolong the blocking time

The antibody concentration may be too high

Use a lower concentration of antibody

Insufficient washing

Increase the frequency and duration of washing

Excessive exposure time

Shorten the exposure time

The membrane or buffer has been contaminated

Use the fresh buffer and keep the membrane moist during the experiment

3. Non-specific bands

Possible causes


The protein sample has digested during the treatment

Choose fresh samples for experiment

The loading amount of sample is too much

Reduce the loading amount

Insufficient blocking

Prolong the blocking time

The washing of membrane may be insufficient

Ensure sufficient washing of membrane

The antibody concentration may be too high

Use a lower concentration of antibody

The antibody specificity is low.

Use antibody with good specificity

The target protein has multiple spliceosomes or modified sites

Refer to relevant literatures to check whether the target protein has other spliceosomes or modified sites

4. Other problems


Possible causes


Black dots on the membrane

The antibodies may have non-specifically binding with the blocking buffer

Replace the blocking buffer

White bands

The target protein content is too high or antibody concentration is too high

Reduce the loading amount or decrease the concentration of the antibody

Molecular weight is very low or high

Inappropriate gel percentage or uneven gel. /The electrophoresis temperature may be too high

Change the gel percentage: use a higher percentage for small proteins and a lower percentage for large proteins

Uneven bands/ Bands trail or deviate or diffuse to both sides

The equipment is not suitable. / There is bubble at the bottom/ The sample is not dissolved well. / The electrode is not balanced. / The sample amount is too much

Ensure that the electrophoresis gel is in good state and horizontal position/ Ensure the sample extraction/ Reduce the sample amount

Summary: The Western blot technology is rather mature, but it is not easy to get the desired result with just one trial. It is recommended to explore and optimize the experimental conditions, thus to get your ideal Western bands in the formal experiment.