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Technique | Advantages | Disadvantages |
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Electron microscopy | Golden standard.Morphological characterization of autophagosomes, autolysosomes and their cargo. | Equipment and expertise required.Difficult to make quantitative analyses. |
Atg8/LC3 conjugation to lipid | Rapid detection and quantification of autophagy.Amenable to high throughput techniques.Used to create transgenic organisms for in vivo study of autophagy. | Dots do not always reflect autophagic activity.Molecular weight shift tests need careful interpretation. |
Other molecular markers (Atg5, Atg6, Atg16 and Atg18 detection) | Detection of various stages of autophagic vesicle formation. | Most of them need further evaluation. |
PI3P detection | Reflects the activity of Vps34 kinase.Quantitative analysis possible. | PI3P accumulation in phenomena not directly related to autophagy (vesicular transport). |
Atg1 and Atg4 activity | Determination of enzymatic activity. | So far no clear kinetic studies were published. |
P62/SQSTM1 degradation | Activated especially by protein aggregates. | Not all stimuli activate its degradation.Orthologue in plants? |
Lysotracker and acridine orange staining | Determination and quantification of autophagy-related lytic activity (lysosomal/vacuolar).FACscan analysis possible. | Autophagosomes are not detected.Lytic activity induced by other conditions as well. |
MDC staining | Determination and quantification of autophagy-related lytic activity (lysosomal/vacuolar). | Not all autophagosomes are detected.Lytic activity induced by other conditions as well. |
Long-lived protein degradation | Measures autophagic degradation of proteins.Kinetic measurements possible. | Nonspecific degradation of proteins by mechanisms other than autophagy. Radioactive technique. |
Sequestration of sugars | Measures autophagic sequestration phase.Quantification may be possible. | Sugars may be metabolized. |
Phosphorylcholine accumulation | Promising plant autophagy technique.Quantification may be possible. | Quantification requires special equipment (NMR spectroscopy). |
Nonselective and selective degradation of proteins | Promising techniques for plant autophagy.Detection of both sequestration and degradation phases.Quantification may be possible. | Autophagy target proteins need further characterization. |
Test of mitophagy or chloroplast autophagy | Detection of autophagy target organelle degradation.Various organelle-specific proteins or organelle-tagged may be used. | Quantification not always possible. |
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