No Signal or Weak Signal (Western Blot (Sigma Protocol))

Question

Accepted Answer

No bands or very faint bands appear on the western blot after detection Common causes: 1) Substrate or conjugate weak or no longer active due to age or improper storage 2) Incorrect dilution of primary or secondary antibody 3) Protein of interest not present or present in low amounts 4) Poor protein transfer from gel to membrane 5) Incubation times inadequate Solutions: 1) Test conjugate and substrate for activity by adding enzyme conjugate to substrate solution 2) Check literature for recommended antibody dilutions and try a range of dilutions 3) Check protein loading amount and consider enriching sample by immunoprecipitation 4) Check protein transfer by staining membrane with Ponceau S prior to blocking 5) Incubate at least one hour with primary antibody