Western blotting is a powerful complex and sensitive technique for quantifying and detecting proteins. The results can be affected by several factors, that also include user error.

Here are a few ways that user error can contribute to variability in western blots:

  • Sample preparation: Western blotting requires careful preparation of the sample, including lysis of the cells, extraction of the proteins, and quantification of the protein concentration.
  • Gel electrophoresis: The gel electrophoresis step is another critical step in western blotting. The type of gel used, the voltage and running time, and the buffer composition can all affect the separation of the proteins and the quality of the transfer to the membrane.
  • Transfer: The type of membrane used, the transfer buffer composition, and the transfer time can all affect the efficiency of the transfer and the quality of the blot.
  • Blocking: Blocking is an important step to prevent nonspecific binding of the antibodies to the membrane. If the blocking step is not done correctly, it can lead to increased background signal and reduced sensitivity.
  • Antibody incubation:  The concentration of the antibodies, the incubation time, and temperature are all critical. Primary antibody and washing step should be conducted at 4 degrees C.
  • Detection: There are a variety of detection methods that can be used for western blotting. The choice of detection method will depend on the type of antibody used and the desired sensitivity. If the detection step is not done correctly, it can lead to inaccurate results.

The Precision Biosystems BlotCycler can reduces operator error in western blotting:

BlotCycler™ automates solution changes, blocking, incubation of primary and secondary antibodies, and washing steps.

  • Eliminate variations in fluid delivery and timing.
  • Get reproducible results, repeatedly.
  • Perform membrane processing precisely, every time.
  • Program your protocol and walk away.
  • Process up to 24 membranes simultaneously
  • Get precise publication-quality results time after time.

 

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