tpp-2 Antibody

Tripeptidyl peptidase II (TPP2) antibodies are critical tools for studying this cytosolic protease's roles in immunology and disease pathogenesis. Below are research-focused FAQs addressing key technical and conceptual challenges:

How do I validate the specificity of a TPP2 antibody for immunoblotting?

• Methodological approach:

  • Use lysates from TPP2-knockdown/knockout cells (e.g., CRISPR-modified cell lines) as negative controls .

  • Compare observed vs. calculated molecular weights (e.g., ~138 kDa observed vs. 104.5 kDa predicted, indicating post-translational modifications) .

  • Validate across species using tissues/cells from human, mouse, and rat .

What are the primary applications of TPP2 antibodies in basic research?

• Key applications:

  • Immunohistochemistry: Detect TPP2 in microglia at active demyelination sites in multiple sclerosis (MS) brain samples .

  • Flow cytometry: Analyze TPP2 expression in immune cell subsets (e.g., CD68+ macrophages, MHC II+ cells) .

  • Functional studies: Correlate TPP2 levels with CD8+ T cell senescence markers (e.g., CD57) in immunodeficiency models .

How do I select an optimal TPP2 antibody for dual-labeling experiments?

• Criteria:

  • Confirm non-overlapping epitopes with co-targets (e.g., CD68, MHC class I) via peptide blocking assays .

  • Prioritize antibodies validated for multiple techniques (e.g., A06668-2: WB, IF, IHC, flow cytometry) .

How to resolve contradictions in TPP2’s role in MHC class I antigen presentation?

• Experimental design:

  • Compare peptide trimming in wild-type vs. TPP2-deficient cells using:

    • Long synthetic peptides (14–17 residues) to assess N-terminal trimming efficiency .

    • Viral challenge models: Measure CD8+ T cell responses to lymphocytic choriomeningitis virus (LCMV) epitopes .

  • Key finding: TPP2 is dispensable for most epitopes but critical for processing extended precursors (e.g., 17-mer peptides) .

What protocols optimize TPP2 detection in activated microglia?

• Technical considerations:

  • Tissue fixation: Use IHC enzyme antigen retrieval (e.g., AR0022 reagent) for formalin-fixed MS brain samples .

  • Antibody dilution: 2–5 μg/mL for paraffin sections, 5 μg/mL for immunofluorescence .

  • Controls: Include periplaque white matter (high TPP2) vs. normal-appearing white matter (low TPP2) .

How to investigate TPP2’s metabolic role in autoimmune models?

• Integrated workflow:

  • Enzymatic assays: Measure TPP2 activity in PBMCs using fluorogenic substrates (e.g., Ala-Ala-Phe-AMC) .

  • Metabolic profiling: Link TPP2 levels to aerobic glycolysis via lactate production assays .

  • Murine models: Compare disease progression in TPP2-deficient vs. wild-type mice under EAE (experimental autoimmune encephalomyelitis) .

Data Contradiction Analysis

Case Study Table: TPP2 Deficiency Phenotypes

Antibody Performance Table