Top 8 reasons for high background noise on your western blot

November 2, 2012 at 1:10 pm Leave a comment

The following are the top 8 causes for overall high background on your western blot and potential solutions:

  1. Blocking was incomplete
    • Increase the concentration of blocker
    • Increase the duration of the blocking step
    • Use a different blocking agent
  2. Blocker was impure
    • Use a pure protein such as BSA or casein as a blocker
  3. Wash protocols were insufficient
    • Increase the number, duration, or stringency of the washes
    • Include progressively stronger detergents in the washes; for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween 20
    • Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding
  4. The blot was left in the enzyme substrate too long (colorimetric detection)
    • Remove the blot from the substrate solution when the signal-to-noise level is acceptable, and immerse in diH2O
  5. Contamination occurred during electrophoresis or transfer
    • Discard and prepare fresh gels and transfer solutions
    • Replace or thoroughly clean contaminated foam pads if a tank blotter was used
  6. Excessive amounts of protein were loadedon the gel or too much SDS was used inthe transfer buffer. Proteins can pass through the membrane without binding and recirculate through a tank blotting system.
    • Reduce the amount of protein on the gel or SDS in the transfer buffer
    • Add a second sheet of membrane to bind excess protein
  7. The primary or secondary antibody was too concentrated
    • Increase antibody dilutions
    • Perform a dot-blot experiment to optimize working antibody concentration
  8. Incubation trays were contaminated
    • Clean the trays or use disposable trays

For more great information, download the protein blotting guide from Bio-Rad Laboratories.

Related Posts:
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Protein blotting guide for novice and advanced users

Entry filed under: Gel Imaging and Western Blots, protein blotting, Western Blot and ELISA. Tags: , , .

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