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 1：The loading amount of sample is low

Suggestions 1：Increase the loading amount

Possible causes 2：The target protein quantity is too low or not expressed in the sample

Suggestions 2：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

Possible causes 3：There is a poor transfer of protein to membrane or not enough protein is bound to the membrane

Suggestions 3：Ensure that the transfer order is correct and the transfer time is sufficient

Possible causes 4：The antibody concentration may be too low

Suggestions 4：Use a higher concentration of antibody

Possible causes 5：The antibody concentration may be too high to cause the signal to disappear instantly

Suggestions 5 : Use a lower concentration of antibody

Possible causes 6：Insufficient exposure time

Suggestions 6：Prolong the exposure time

Possible causes 7：Insufficient incubation or deactivation of the substrate

Suggestions 7：Increase the incubation time of substrate and ensure that the substrate is valid

Possible causes 8：The target protein transferred to the membrane has degraded

Suggestions 8：Keep a low transfer temperature, decrease the transfer electric current and transfer time

Possible causes 9：Excessive washing of the membrane

Suggestions 9：Reduce the frequency or duration of washing steps

Possible causes 10：The antibody is inactive or the titer of antibody is too low

Suggestions 10: Pay attention to the preservation of antibody and use antibody with higher titer

1. High background

Possible causes 1：The experimental equipment has been contaminated

Suggestions 1 : Ensure that the equipment is clean

Possible causes 2：Some membrane may cause high background

Suggestions 2 : NC membranes are considered to cause less background than PVDF membranes.

Possible causes 3：Blocking buffer is not compatible or there is cross-reactivity between the blocking buffer with antibodies

Suggestions 3: Replace the blocking buffer

Possible causes 4：Insufficient blocking

Suggestions 4: Prolong the blocking time

Possible causes 5：The antibody concentration may be too high

Suggestions 5 : Use a lower concentration of antibody

Possible causes 6：Insufficient washing

Suggestions 6: Increase the frequency and duration of washing

Possible causes 7：Excessive exposure time

Suggestions 7: Shorten the exposure time

Possible causes 8：The membrane or buffer has been contaminated

Suggestions 8: Use the fresh buffer and keep the membrane moist during the experiment

1. Non-specific bands

Possible causes 1: The protein sample has digested during the treatment

Suggestions 1: Choose fresh samples for experiment

Possible causes 2: The loading amount of sample is too much

Suggestions 2: Reduce the loading amount

Possible causes 3: Insufficient blocking

Suggestions 3: Prolong the blocking time

Possible causes 4: The washing of membrane may be insufficient

Suggestions 4: Ensure sufficient washing of membrane

Possible causes 5: The antibody concentration may be too high

Suggestions 5: Use a lower concentration of antibody

Possible causes 6: The antibody specificity is low.

Suggestions 6: Use antibody with good specificity

Possible causes 7: The target protein has multiple spliceosomes or modified sites

Suggestions 7: Refer to relevant literatures to check whether the target protein has other spliceosomes or modified sites

1. Other problems

Problem 1: Black dots on the membrane

Possible causes 1: The antibodies may have non-specifically binding with the blocking buffer

Suggestions 1: Replace the blocking buffer

Problem 2: White bands

Possible causes 2: The target protein content is too high or antibody concentration is too higSuggestions 2: Reduce the loading amount or decrease the concentration of the antibody

Problem 3: Molecular weight is very low or high

Possible causes 3: Inappropriate gel percentage or uneven gel. /The electrophoresis temperature may be too high

Suggestions 3: Change the gel percentage: use a higher percentage for small proteins and a lower percentage for large proteins

Problem 4: Uneven bands/ Bands trail or deviate or diffuse to both sides

Possible causes 4: 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

Suggestions 4: 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.