Western Blot Troubleshooting

Regardless of how well you know how to do a western blot, you’ll inevitably run into trouble. It happens to the best of us, and can be very frustrating when we need reliable western blots to gather data. This troubleshooting guide is designed to prevent, or quickly solve, any problems you may have with your western blotting technique.

Weak or No Signal
  • ECL is not working: Try a new bottle of ECL or a different brand. Try to develop a blot with an antibody that is known to work.
  • Developer is not working: Try a different developer in your research institute.
  • Protein of interest is not expressed: It’s possible your protein of interest is not expressed in your cell, or is expressed at very low levels. Use a known positive control to check.
  • Bad primary antibody: Your primary antibody may have low affinity for your protein of interest. Try a different antibody to the same protein (Check other options first, as antibodies are expensive).
  • Bad secondary antibody: Your HRP-conjugated secondary antibody may be dead. Check with a known primary antibody, or order new secondary antibody.
  • Transfer was ineffective: In order to have a strong signal, the transfer from the gel to the blot must be effective. Stain the gel with coomassie after transfer to examine how much protein is left.
High Background
  • Insufficient washing: Washing should be thorough at each step with TBST. Be sure to use at least 25 ml of TBST for each wash step to ensure the  blot is clean. Also be sure to use clean trays to ensure antibodies are not carried over between washes.
  • Poor blocking: The blocking agent may be ineffective for your protein-antibody combination. Try a different blocking agent.
  • Blocking agent cross-reacts with antibody: It’s possible that the primary antibody actually cross-reacts with your blocking agent. This will give an extremely high background. Try a different blocking buffer if you think you may have this problem. Milk is known to cross-react with certain phospho-specific antibodies because the milk protein casein is a phospho-protein.
  • Antibody incubation temperature is too high: Generally, incubating your western blot at room temperature will work just fine. However, sometimes incubating at 4°C will reduce background. We recommend doing all western blot antibody incubations at 4°C to be safe.
  • PVDF membrane was allowed to dry: If the PVDF membrane is allowed to dry at any time after methanol activation, it will give a high background. Avoid letting the PVDF dry at all costs.

Non-Specific Bands
  • Bad Primary Antibody: It’s possible that your primary antibody cross-reacts with other proteins in the cell lysate being western-blotted. You may have to try a different antibody.
  • Blocking agent was ineffective: The correct blocking agent must be used at a sufficiently high concentration to have a clean western blot. If you have multiple, non-specific bands, the first thing you may want to change is your choice of blocking agent. Alternatively, you can try to increase the concentration of the blocking agent.
  • Post-translation modification: The protein recognized by the antibody may be modified in cells. If the bands look like doublets, or you can decipher a distinct relationship (e.g. cleavage product), the multiple bands may be the same protein in different forms.
Speckled or Dotted Appearance of Film
  • Black Spots: The membrane is dirty or there is undissolved blocking agent in the buffer.
  • White Spots: Air bubbles were trapped at some point during the western blotting process. This could happen during protein transfer or development in the film cassette.
Uneven Bands (Circles at corners, or smiling)
  • This is most likely due to excessive heating of the gel during electrophoresis. Try running the gel in an ice bucket or in a cold room at 4°C.
Thick Bands
  • Stacking was ineffective: In order to stack effectively, the height of the stacking gel must be at least 100% the height of the wells into which the lysate is loaded. A stacking gel that is 1.5 times the height of the wells is better.

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