How to perform subsequent or ‘double’ immunostaining of two different antigens on a single nitrocellulose blot within one day with and immunoperoxidase technique

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Abstract
Defining the properties of antibody preparations has been facilitated by immunoblotting techniques: by ‘double’ immunostaining of blots, however, one can clearly visualize the properties of two (or even more) antibodies on a single blot. We describe a one‐day procedure in which two different antisera, both raised in rabbits, can subsequently be applied to a single nitrocellulose blot of protein samples separated by either native or sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis. In order to visualize antibody activity, a three‐step unlabeled immunoezyme method ‐ the peroxidase anti‐peroxidase technique ‐ has been used for both first and second immunostaining. Discrimination between the results of each of the immunostainings has been achieved by using 3,3′ ‐diaminobenzidine and 4‐chloronaphtol as different peroxidase substrates which yield differently colored precipitates. The problem of method overlapping has been overcome by developing a short but efficient elution procedure for the elution of antibody bound to the blot during the first immunostaining: going from ‘mild’ conditions (suited for incubations with sera) to ‘denaturing’ conditions (low pH, low ionic strength, 20 % dimethylformamide) and vice versa repeatedly does not affect antigens but elutes antibodies completely.