tpst-2 Antibody

Here’s a structured FAQ collection for researchers working with TPST-2 antibodies, categorized into basic and advanced research questions, with methodological guidance and data-driven insights:

How do I validate the specificity of TPST-2 antibodies in Western blotting?

Methodological Answer:

• Use knockout (KO) controls (e.g., Tpst2−/− mouse tissue lysates) to confirm antibody specificity. Compare band patterns between wild-type and KO samples. For example, a 31-kDa band observed in wild-type epididymis lysates disappears in Tpst2−/− samples, confirming specificity .

• Include peptide blocking assays: Pre-incubate the antibody with its immunizing peptide to verify signal loss.

What are the optimal applications for TPST-2 antibodies in protein sulfation studies?

Key Applications:

How to select TPST-2 antibodies for cross-species reactivity?

• Prioritize antibodies validated in multiple species (e.g., rabbit polyclonal RB4954 reacts with human, mouse, rat, cow, and chicken TPST-2) .

• Confirm ortholog homology: TPST-2 shares >80% sequence similarity between humans and mice .

How to design experiments assessing TPST-2’s role in bacterial toxin susceptibility?

Stepwise Approach:

• Knockdown Models: Use shRNA (e.g., TPST2-targeted lentivirus) in THP-1 or RAW264.7 cells .

• Functional Readouts:

  • Measure leukocidin cytotoxicity via LDH release assays (e.g., 20 nM HlgCB reduces viability by 70% in controls vs. 20% in TPST2-KD cells) .

  • Quantify receptor sulfation via flow cytometry (e.g., anti-sulfotyrosine antibodies detect C5aR1 modification) .

• In Vivo Validation: Compare Tpst2−/− myeloid cells with wild-type in murine S. aureus infection models .

How to resolve contradictions in TPST-2’s association with disease pathways?

Case Example:

• Chronic Pancreatitis: No association found between TPST2 variants (e.g., p.R153H) and disease risk in sequencing studies .

• Inflammatory Disorders: TPST-2 mediates leukocidin cytotoxicity by sulfating phagocyte receptors (e.g., C5aR1), exacerbating S. aureus infections .
  Resolution Strategy:

• Context-dependent analysis: Tissue-specific sulfation patterns (e.g., epididymal vs. immune cells) explain divergent roles .

What controls are critical for TPST-2 functional studies in transfected cells?

Essential Controls:

Why do some studies report TPST-2 as non-essential in pancreatitis despite its enzymatic activity?

• Key Findings:

  • TPST2 variants (e.g., c.458G>A) retain full sulfotransferase activity in vitro .

  • Functional redundancy: TPST1 compensates for TPST2 loss in certain tissues .

• Experimental Adjustment: Include dual Tpst1/Tpst2 KO models to assess redundancy.

How to optimize TPST-2 detection in low-abundance samples?

• Signal Amplification: Use tyramide-based IHC amplification for tissue sections .

• Fraction Enrichment: Isolate Golgi membranes via differential centrifugation .

Mechanistic Insights from Recent Studies