Evaluation of Double Immunolabeling Methods for Epitope Mapping on Isolated Na, K-Atpase Membranes

Epitopes of the Na, K-ATPase a subunit were mapped on isolated, unfixed plasma membranes by double immunolabeling electron microscopy with protein A gold and negative staining. In this study we have defined several factors, which influence the labeling of the epitopes with sequence-specific antibodies. The results demonstrate that simultaneous labeling with low and high affinity antibodies can be achieved with a labeling sequence consisting of low affinity antibody―small gold probe―high affinity antibody―large gold probe, and that incubation with free protein A is essential for masking cross-contamination caused by attachment of the second probe to unoccupied Fc portion of the first antibody. Equal labeling was obtained on membranes without fixation and after fixation with 4% formaldehyde. An estimate of labeling efficiency showed that 1.3% of the total a subunit N-termini in p21 Na, K-ATPase membrane crystals could be detected with a 5 nm gold probe. The present results define optimal conditions for the application of double immunonegative staining to isolated membranes.