Western blotting is a standard laboratory tool for detecting and quantifying specific proteins in a sample. Due to the number of steps involved, it is important to be aware of the common errors that can occur, which can lead to misleading results.

The following are typically the most common errors in Western blotting protocol:

  • Incorrect protein loading: It is important to optimize the amount of protein loaded for each sample and antibody. Loading too much or too little protein onto the gel can lead to a variety of problems, including nonspecific bands, weak signals, or saturated bands.
  • Improper blocking: Blocking is an essential step in Western blotting to prevent nonspecific binding of antibodies to the membrane. The detection method being used should be compatible with the blocking buffer. Problems can arise if blocking buffer interferes with the detection method. If you are unsure about compatibility a preliminary test should be done. Also using too much blocking buffer or an inappropriate blocking agent can also lead to nonspecific bands.
  • Overexposure of the blot: It is important to optimize the exposure time for each blot to achieve the best results Overexposing the blot can result in saturated bands and a high background.
  • Incorrect antibody dilution: It is important to optimize the antibody dilution for each experiment. Antibody titration using the Blotcycler touch automates the titration process in one simple step. Using too much or too little primary or secondary antibody can lead to a number of problems, including nonspecific bands, weak signals, or saturated bands.
  • Insufficient washing or washing at RT: All washing steps should be done at 4 degrees Celsius. It is important to note that the optimal number of washing steps may vary depending on the specific antibody used, the concentration of the antibody, and the sensitivity of the detection method. It is therefore important to experiment with different washing protocols to find the optimal number of washes for each antibody. The BlotCycler automates this process which leads to  the precise timing of each wash cycle. After each incubation step, it is important to wash the blot thoroughly to remove unbound antibodies and other reagents. Insufficient washing can lead to nonspecific bands and a high background.

Other mistakes that will cause problems:

  • It is important to use fresh, quality reagents.
  • Avoid air bubbles during blotting, this can prevent the proteins from transferring evenly.
  •  Improper transfer can lead to a variety of problems, including uneven transfer, partial transfer, or no transfer at all.
  • Make sure your detection method is correct and suitable for the antibody being used.

Many of these issues can be avoided by automating as many steps as possible. Both the BlotCycler Touch or Blotcycler Mini will automate all the Blot processing steps resulting in reproducible publication quality results.