All lanes : Anti-14-3-3 Tau antibody (ab52834) at 1/1000 dilutionLane 1 : a total protein extract from E coli with 50ng to 100 ng of a tagged fusion protein of an irrelevant antigenLane 2 : a total protein extract from E coli with 50ng to 500ng of the antigen (tagged fusion protein)Lysates/proteins at 20 µg per lane.SecondaryLane 1 : Rabbit anti-mouse IgG + IgM, (H+L) horseradish peroxidase conjugated, at 1/5000 dilutionLane 2 : Rabbit anti-mouse IgG + IgM, (H+L) horseradish peroxidase conjugated, at 1/5000 dilution