Loss of antigens from immunoblotting membranes

Immunoblotting of PAGE-separated proteins is widely used. After electrophoretically transferring proteins from PAGE gels, sites on the membrane which are unoccupied are blocked or quenched with excess protein. The sensitivity of the immunoblotting technique is in part dependent on the amount of antigen associated with the membrane. We have investigated the loss of blotted proteins during blocking of various membranes with milk. Transblotted proteins are lost from nitrocellulose membranes (NC) (0.45 microns) in proportion to the amount of milk in the blocking solution and loss increases with time. When 5% milk was used as the blocking solution the amount lost was about 10% within 1 h and approached 25% at 24 h. This loss was nonselective and involved blotted antigens of a wide range of Mr. This time- and concentration-dependent relationship for the loss of transblotted proteins was tested with four immunoblotting membranes: Immobilon-P (Millipore), NitroPlus-2000 (MSI), and NC (0.2 microns and 0.45 microns pore size). NC (0.2 microns) gave the best results with the lowest percentage loss at 1 h (less than 5%). We recommend using NC (0.2 microns) and blocking with 0.5% milk for 30 min to minimize the loss of antigens from immunoblotting membranes.