Page 1 of 7
This is a detailed troubleshooting for western blotting.
No signal
The primary antibody and the secondary antibody are not compatible.
Use secondary antibody that was raised against the species in which the primary was raised (e.g primary is aised in rabbit, use anti-rabbit secondary).
Not enough primary or secondary antibody is bound to the protein of interest.
Use more concentrated antibody. Incubate longer (e.g. overnight) at 4ºC.
Cross-reaction between blocking agent and primary or secondary antibody.
Use a mild detergent such as Tween20 or switch blocking reagent (i.e. commonly used blocking reagents re milk, BSA, serum or gelatin).
The primary antibody does not recognize the protein in the species being tested.