Specific detection of particular antigens or haptens

The protein used for quenching is mostly BSA (3%) in 10 mM Tris-HCl buffer, pH 7.4, containing 0.9% NaCl, for 1 h at 40°C (Towbin et al., 1979) or 0.5% for 24 h at room temperature (Erickson et al., 1982), but ovalbumin, hemoglobin or gelatin can also be used (White et al., 1981 Gershoni and Palade, 1982 Lee et al., 1982). After quenching, the membranes are rinsed with Tris-buffered saline. 

Detection of antigens on nitrocellulose (adapted from Batteiger et al., 1982  

Incubate with primary antibodies (3 h at room temperature, overnight for high dilutions). Antiserum dilutions are made in blocking solutions. 

Wash for 30 min with several changes of TBS and repeat step 2. 

Incubate with the conjugate in blocking solution (2 h about 2 pg conjugate per ml). 

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