At3g12260 Antibody

Here’s a structured collection of academically oriented FAQs for researchers working with the At3g12260 Antibody, derived from scientific literature and technical specifications:

How to validate At3g12260 antibody specificity in Arabidopsis mitochondrial studies?

• Method: Perform Western blot using mitochondrial extracts from wild-type (WT) and At3g12260 knockout mutants. Expected band at 15 kDa in WT lysates (absent in knockouts) confirms specificity .

• Controls: Include a peptide-blocking experiment (pre-incubate antibody with immunogen peptide) to eliminate signal .

• Cross-reactivity: Test species listed in PHY0535A specificity data (e.g., Brassica napus, Zea mays) to assess non-target binding .

What protocols optimize At3g12260 antibody performance in Western blot?

• Dilution range: Start at 1:1000–1:2000 in 5% non-fat milk/TBST .

• Blocking: Use 5% BSA for high-sensitivity applications.

• Detection: Chemiluminescent substrates (e.g., ECL Prime) enhance low-abundance protein detection.

How to resolve discrepancies between observed and predicted molecular weights (MW) in Western blots?

• Scenario: Observed band ≠ 15 kDa .

• Troubleshooting:

  • Post-translational modifications: Test lysates with phosphatase/protease inhibitors.

  • Alternative splicing: Validate transcript variants via RT-PCR.

  • Artifacts: Run SDS-PAGE with high-resolution markers (e.g., 10–250 kDa gradient gels).

Can At3g12260 antibody differentiate between mitochondrial Complex I subunits and biogenesis factors?

• Experimental design:

  • Isolate mitochondrial complexes via BN-PAGE (Blue Native PAGE) .

  • Probe with At3g12260 antibody.

  • Co-stain with known Complex I markers (e.g., NADH dehydrogenase subunits).

• Interpretation: Co-migration with markers indicates subunit role; distinct bands suggest biogenesis factor .

How to interpret weak/no signal in cross-species applications?

• Approach:

  • Phylogenetic alignment: Compare At3g12260 epitope region (e.g., residues 50–100) across species .

  • Empirical testing: Use lysates from species in PHY0535A specificity panel .

• Solution: Request custom peptide affinity purification for non-Arabidopsis species .

What strategies mitigate batch-to-batch variability in long-term studies?

• Standardization:

  • Aliquot reconstituted antibody to avoid freeze-thaw cycles .

  • Include a reference sample (e.g., WT mitochondrial extract) in every blot.

• Validation: Compare new batches with existing stocks using quantitative densitometry.

Table 1: Cross-reactivity of At3g12260 Antibody

Table 2: Antibody Stability Under Storage Conditions