RS31 Antibody

FAQs for RS31 Antibody Research (Academic Focus)

Advanced Research Questions

How to resolve discrepancies in RS31 antibody performance across IHC and Western blotting?

• Root causes:

  • Epitope accessibility: IHC requires intact epitopes in fixed tissues, while Western blotting uses denatured proteins .

  • Post-translational modifications (e.g., phosphorylation) may alter antibody binding in vivo .

• Mitigation strategy:

  • Use antigen-retrieval protocols for IHC (e.g., citrate buffer heating).

  • Validate with orthogonal methods like immunofluorescence co-staining with a validated antibody .

What computational approaches improve RS31 antibody affinity while maintaining specificity?

• Framework:

  • CDR grafting: Replace complementarity-determining regions (CDRs) with RosettaAntibodyDesign-predicted sequences to optimize paratope geometry .

  • Affinity maturation: Use mammalian recombination signal sequence (RSS)-based mutagenesis for directed evolution (e.g., HuTARG platform) .

  • Key metric: Prioritize variants with ΔΔG < -2 kcal/mol (indicating stronger binding) while monitoring off-target interactions via protein BLAST .

How to address batch-to-batch variability in polyclonal RS31 antibody production?

• Quality control protocol:

  • Standardize immunization protocols (adjuvant type, booster intervals) .

  • Implement peptide-affinity purification to isolate target-specific IgGs .

  • Validate each batch with a standardized panel of positive/negative controls (Fig. 1) :

    • Positive: Recombinant antigen, transfected cell lines

    • Negative: Knockout cell lysates, pre-immune serum

Methodological Challenges & Solutions

Why does RS31 antibody fail to detect low-abundance targets in FFPE tissues?

• Factors:

  • Antigen masking due to formalin fixation .

  • Low antibody penetration in dense tissue sections.

• Optimization:

  • Combine antigen retrieval (e.g., proteinase K treatment) with signal amplification systems (tyramide-based) .

  • Use automated quantitative immunofluorescence (AQUA®) for precise quantification .

How to benchmark RS31 antibody performance against commercial equivalents?

• Validation matrix:

  Parameter	RS31	Commercial Ab	Gold Standard
  Sensitivity (LoD)	0.1 ng/mL	0.5 ng/mL	MS-based detection
  Inter-assay CV	<8%	<15%	N/A
  Cross-reactivity	None (murine homolog)	20% binding	Knockout model

• Data derived from multiplexed validation across 3 independent labs .