Nitrogenase quantification
| What is being measured in 1 - 3 words |
|---|
| Approach: immunolabelling |
| Context: in situ or enviroment, incubation |
| Spatial scale: |
| Temporal scale: days, seasons |
| Units: signal intensity, counts |
| Community captured: nitrogenase-expressing diazotrophs |
| Co-measurements: |
Method Overview
Nitrogenase quantification by immunolabelling targets nitrogenase protein subunits using specific antibodies against the NifH protein. Cells are fixed and incubated with primary antibodies that bind nitrogenase, followed by enzyme-labeled secondary antibodies for detection. Immunolabelling can be combined with epifluorescence microscopy, confocal microscopy, or flow cytometry to quantify the presence, abundance, and spatial distribution of nitrogenase-expressing diazotrophs in environmental samples. This approach provides a direct measure of nitrogenase protein presence rather than gene abundance or enzymatic activity.
Output
- Presence and abundance of nitrogenase-positive cells
- Spatial distribution of nitrogenase-expressing diazotrophs
- Relative protein expression levels (method-dependent)
Scale of measurement
- Protein / cellular level
- Semi-quantitative
- Single-cell to community-level inference
Data generated
- Fluorescence microscopy images
- Flow cytometry counts (if applicable)
- Signal intensity measurements
- Cell abundance estimates linked to nitrogenase expression
Units & currency
- Fluorescence intensity (relative units)
- Counts of nitrogenase-positive cells
- Proportion of labeled cells (%)
Sample size
- Varies by study design
- Includes biological and technical replicates
Repositories & databases
- No centralized database for nitrogenase immunolabelling data
Limitations
- Requires highly specific and validated antibodies
- Signal intensity can vary with fixation and staining conditions
- Limited taxonomic resolution without complementary methods (e.g., FISH)
- Antibody availability is biased toward well-studied diazotrophs
Example Applications & Protocols
Classic examples
Recent applications
Protocols:
- Immunofluorescence staining protocols targeting NifH
- Microscopy-based quantification workflows
- Custom laboratory SOPs optimized for fixation and antibody specificity
Common calculations/conversions
- Percentage of nitrogenase-positive cells = (labeled cells / total cells) × 100
- Normalization of signal intensity across samples
- Integration with cell counts for community-level estimates