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ND3 redox state (Click FLAG (Advantages (PEG free, Bio-orthogonal, Success…
ND3 redox state
Click FLAG
Advantages
PEG free
Bio-orthogonal
Success can be assessed with pan-FLAG antibody
Rapid
Immunological
Disadvantages
2 different chemical approaches
Expensive
PEG
Disadvantages
Requires PEG
Only works on lysate
Mass shift limited by acceptable PEG size
Could be sterically hindered
Advantages
Biotin step could be added for in gel.
Unlikely to interfere with analysis
One step
Could work on isolated mitochondria
Flexible
Time and cost efficient
Immunological
Mitochondria
Disadvantages
No current lysis method
Additional time
Advantages
Literature contains methods
Enables de-active transition to be assessed
Only acceptable starting point for follow-up analysis
Enrichment
Redox blot
Advantages
Simple
Immunological
Time and cost efficient
Ratio-metric
Flexible
Disadvantages
Unsure what the upper and lower bands correspond to
Only probe reduced versus oxidised
Requires modifications
Requires modification to assess solvent exposed
Interpretation required to understand why the mass shift occurs--must be to do with free cysteine
Co-IP
Disadvantages
Time consuming
Antibody may be unsuitable for Co-IP
Intensity based between samples
Low-throughput
Normalising protein at low amounts
Need for pH corrections
Advantages
Assay can be done in-gel
Amenable to fluorescent NEM
Clean
Clickable
Dot blot
Advantages
High throughput
Minimal material required
Immunological
Mass shift free
Clickable
Disadvantages
Requires enriching fractions
May require additional equipment and reagents
Normalising protein at low amounts