Common Western blotting mistakes that can lead to inaccurate results:

  • Using the wrong antibody. The antibody is the most important reagent in a Western blot, so it is essential to use the right antibody for the target protein. If the antibody is not specific for the target protein, it will bind to other proteins in the sample, leading to false positive results. The primary antibody step should be done at 4 degrees Celsius.
  • Using too much or too little antibody. The amount of antibody used can also affect the results of a Western blot. If too much antibody is used, it can saturate the binding sites on the membrane, leading to a decrease in sensitivity. If too little antibody is used, it may not be able to bind to the target protein, leading to false negative results. It’s a good ideal to titrate your antibody to find the correct concentration; the titration procedure can be automated using the BlotCycler Touch.
  • Not blocking the membrane properly. Blocking the membrane is essential to prevent non-specific binding of the antibody. If the membrane is not blocked properly, the antibody may bind to the membrane instead of the target protein, leading to false positive results. Washing at 4 degrees Celsius is recommended.
  • Not washing the membrane properly. Washing the membrane removes unbound antibody and other contaminants. If the membrane is not washed properly, it can lead to high background signal and decreased sensitivity. It is best to determine the optimal number of wash steps for each antibody. All wash steps can be automated using the BlotCycler Touch or Mini.
  • Using the wrong detection reagent. The detection reagent is used to visualize the bands on the Western blot. If the wrong detection reagent is used, it may not be able to detect the target protein, leading to false negative results.
  • Not following the protocol properly. It is important to follow the protocol carefully for each step of the Western blot procedure. If any steps are skipped or done incorrectly, it can lead to inaccurate results. In order to generate reproducible results the timing of each step should be as exact as possible. The entire blot processing procedure can be automated using the BlotCycler ensuring reproducible and quantifiable results.

Remember, for accurate and reliable results:

  • Use a high-quality antibody that is specific for the target protein.
  • Use the correct amount of antibody.
  • Block the membrane properly.
  • Wash the membrane properly.
  • Use the correct detection reagent.
  • Follow the protocol carefully.
  • Practice the technique regularly.

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