ACT11 Antibody

How is the specificity of ACT11 antibody validated in plant actin studies?

The ACT11 antibody (mAb2345a) is validated using:

• Western Blot (WB): Detects a single band at ~45 kDa in Arabidopsis lysates, aligning with ACT11's molecular weight .

• Immunofluorescence (IF): Localizes to cytoskeletal structures in plant tissues, consistent with actin's role .

• Cross-reactivity testing: No reactivity observed with non-plant actins (e.g., mammalian or fungal) .

Table 1: Validation Data for ACT11 Antibody

What experimental controls are critical when using ACT11 antibody in immunoassays?

• Negative controls: Use tissue/cell lysates from ACT11-knockout mutants.

• Isotype controls: Mouse IgG1 to rule out nonspecific binding .

• Competition assays: Pre-incubate antibody with purified ACT11 antigen to confirm signal loss .

How does ACT11 antibody distinguish between vegetative and pollen actin subclasses?

The antibody recognizes epitopes in Thr43 (plant actin) and Thr41 (Dictyostelium), enabling subclass-specific detection:

• Vegetative actins: Binds subclasses 2 and 3 via conformational epitopes .

• Pollen actins: Targets subclasses 4 and 5 due to post-translational modifications (e.g., phosphorylation) .

Advanced Research Questions

How can researchers resolve discrepancies in ACT11 antibody reactivity across plant species?

• Epitope mapping: Compare ACT11 sequences to identify conserved regions (e.g., Thr43) .

• Peptide blocking: Synthesize Thr43-containing peptides to test cross-reactivity .

• Structural analysis: Use cryo-EM or X-ray crystallography to assess antibody-antigen binding dynamics .

What methodologies optimize ACT11 antibody performance in low-abundance protein detection?

• Signal amplification: Pair with tyramide-based systems in IF .

• Pre-clearing lysates: Remove nonspecific proteins via protein G beads .

• Multiplex assays: Combine with antibodies against co-expressed markers (e.g., tubulin) for dual staining .

Table 2: Troubleshooting ACT11 Antibody Sensitivity Issues

How do glycosylation patterns affect ACT11 antibody binding in plant stress responses?

• Enzymatic deglycosylation: Treat lysates with PNGase F to assess glycan dependency .

• LC-MS/MS: Compare glycopeptide profiles under stress vs. control conditions .

• Functional assays: Corrogate actin polymerization rates with antibody binding efficiency .

What computational tools predict ACT11 antibody-antigen binding affinities?

• Molecular docking: Use tools like Rosetta or HADDOCK to model Thr43 interactions .

• Machine learning: Train models on plant actin epitope databases to predict cross-species reactivity .

Methodological Recommendations

• For epitope characterization: Combine alanine scanning mutagenesis with surface plasmon resonance (SPR) .

• For longitudinal studies: Validate antibody stability in long-term storage (-20°C in 0.05% sodium azide) .