Abstract
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.
Original language | English (US) |
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Pages (from-to) | 347-356 |
Number of pages | 10 |
Journal | ACTA HISTOCHEMICA ET CYTOCHEMICA |
Volume | 27 |
Issue number | 4 |
DOIs | |
State | Published - 1994 |
All Science Journal Classification (ASJC) codes
- Pathology and Forensic Medicine
- Biochemistry
- Physiology
- Histology
- Cell Biology