Usemoreconcentratedantibody.Incubatelonger(e.g.overnight)at4ºC.
Useanenrichmentsteptomaximizethesignal(e.g.preparenuclearlysatesforanuclearprotein,etc.).
Donotoverwashthemembrane.
Toomuchblockingdoesnotallowyoutovisualizeyourproteinofinterest.
Insteadofusing5%milkintheantibodybufferstryremovingthemilkorusing0.5%.Switchblockingreagentsorblockforlesstime.