Anti-Cystatin-11 Rabbit Polyclonal Antibody (CST11) for Protease Inhibitor Pathway Research – RUO

Target overview: Cystatin-11 (CST11)

Cystatin-11 (gene symbol CST11) is a member of the type-2 cystatin family (cysteine protease inhibitors) involved in modulating cathepsin activity and proteostasis in specialized tissues. For genomic context and aliases, see NCBI Gene query for CST11 (human) and homologs across species (ortholog lookups, transcripts, and exon/intron maps) via NCBI Gene and UCSC Genome Browser. Curated transcripts and coding sequences for assay design are available in RefSeq and the Consensus CDS (CCDS) project. For protein-level entries (isoforms, predicted signal peptides, and length variants), consult NCBI Protein and conserved domain annotations in NCBI CDD.

Transcript/protein identifiers and variant annotations can be cross-checked using dbSNP, ClinVar (database metadata only; for research use), and structural resources (if available) in NCBI Structure. For expression datasets and sample-level evidence, review GEO and literature indexed in PubMed.

Antibody description

• Product name: Anti-Cystatin-11 Rabbit pAb (polyclonal)

• Host / isotype: Rabbit / IgG (polyclonal)

• Clonality: Polyclonal (multi-epitope binding for robust detection)

• Immunogen: Recombinant fragment or synthetic peptide derived from a conserved region of human CST11 (precise aa range provided on COA). Epitope selection guided by conserved-domain mapping in NCBI CDD and low-homology windows identified using NCBI BLAST.

• Purification: Antigen-affinity purified.

• Applications (RUO): WB, IHC-P, IF/ICC, IP, ELISA capture/detection.

• Species reactivity (predicted/validated): Human (validated). Cross-reactivity to closely related primates may be predicted in silico via Primer-BLAST and Taxonomy sequence checks; empirical verification recommended.

• Recommended dilutions (starting ranges):

  • WB: 1:1,000 – 1:5,000

  • IHC-P: 1:100 – 1:400

  • IF/ICC: 1:200 – 1:800

  • ELISA: 1:5,000 – 1:20,000 (depending on plate and coating)

• Storage/handling: Aliquot and store at −20 °C to −80 °C. Avoid repeated freeze–thaw. For image analysis standardization, NIH ImageJ provides densitometry and fluorescence quantitation workflows.

• Validation framework: Designed and reported under RUO guidelines aligned with NIH Rigor & Reproducibility recommendations; consider referencing metrological concepts from NIST antibody reference materials for internal controls.

Technical background and epitope strategy

Cystatin-11 belongs to the type-2 cystatin superfamily, characterized by a signal peptide, secretion, and a conserved inhibitory wedge that engages papain-like cysteine proteases. Epitope selection avoided highly conserved inhibitory motifs to reduce cross-reactivity with paralogs (e.g., CST1, CST2). In-silico analyses are performed by: (i) domain alignment in CDD, (ii) low-complexity filtering and homology checks via BLASTP, and (iii) transcript consistency across CCDS/RefSeq (CCDS; RefSeq). Genomic coordinates and exon–intron structures can be inspected in UCSC hg38, with In-Silico PCR to visualize amplicon spans for qPCR orthogonal assays.

Applications and experimental notes (RUO)

 Western Blot (WB)

• Sample types: Lysates from tissues/cell lines with documented CST11 expression (screen GEO profiles via GEO).

• Lysis: RIPA or NP-40 with protease inhibitors; quantify by BCA.

• Load: 10–40 µg total protein per lane; SDS-PAGE under reducing conditions.

• Transfer & block: PVDF, 0.2–0.45 µm; 5% BSA or milk.

• Primary incubation: Anti-CST11 Rabbit pAb at suggested dilution; overnight 4 °C recommended.

• Quantitation: Use NIH ImageJ for band densitometry.

• Sequence support: Validate band size against NCBI Protein isoform lengths and signal peptide cleavage predictions; check potential variants in dbSNP and ClinVar (research database context only).

 Immunohistochemistry, paraffin (IHC-P)

• Antigen retrieval: Citrate pH 6 or Tris-EDTA pH 9; optimize by tissue.

• Controls: Include peptide-blocked control when available.

• Imaging: Standardize exposure; capture raw 16-bit where possible; analyze ROIs in ImageJ.

• Expression reference: Cross-reference tissue expression hints from PubMed and sample meta-data in GEO.

 Immunofluorescence / ICC

• Fixation/permeabilization: PFA 4% then Triton X-100 0.1–0.3% (optimize).

• Mounting: Antifade reagent; include nuclear counterstain.

• Colocalization planning: Verify organelle markers against MeSH terminology for consistent reporting.

 ELISA (capture/detection)

• Coating: 0.5–2 µg/mL capture Ab; HRP-conjugate for detection if available.

• Dynamic range: Optimize with serial dilutions and standard curve fitting; document plate layout and raw OD in NIH rigor guidelines-aligned worksheets.

 IP (Immunoprecipitation)

• Beads: Protein A/G magnetic beads; confirm epitope accessibility.

• Orthogonal validation: Post-IP WB against CST11; confirm peptide competition where feasible.

In-silico resources for assay design

• Gene & transcripts: NCBI Gene

• Reference sequences: RefSeq

• Protein records: NCBI Protein

• Conserved domains: NCBI CDD

• Literature: PubMed

• Expression datasets: GEO

• Variant info: dbSNP, ClinVar

• Genomic browser: UCSC hg38

• In-silico PCR: UCSC hgPcr

• BLAST alignment: NCBI BLAST

• Primer design: Primer-BLAST

• Taxonomy context: NCBI Taxonomy

• Structure (if available): NCBI Structure

• Reproducibility recommendations: NIH Rigor & Reproducibility

• Antibody metrology concepts: NIST mAb References

• Genetics education context: NHGRI Genome.gov

• Project search (funding metadata): NIH RePORTER

Antibody validation & QC (reporting template)

• Specificity: Peptide-blocking WB or IHC control; single major band at expected MW (consult NCBI Protein).

• Sensitivity: Titration curve across recommended dilution range; record exposure settings; quantify with ImageJ.

• Orthogonality: qPCR of CST11 transcripts (primers designed via Primer-BLAST) correlated with protein signal.

• Reproducibility: Include biological and technical replicates, pre-registered analysis plan aligned to NIH Rigor guidance.

• Documentation: Report catalog ID, lot number, storage, and complete method steps (buffers, pH, temperature, timings) in lab notebooks referencing MeSH terms for consistent indexing.

Datasheet-style specifications (for your page)

• Form: Liquid, PBS with stabilizers; preservative details on label.

• Concentration: Lot-specific (A280).

• Reactivity: Human (validated); predicted primate cross-reactivity (sequence homology checks via BLAST).

• Application notes: WB, IHC-P, IF/ICC, ELISA, IP (RUO).

• Positive controls (examples): Tissues/cell lines with CST11 expression per GEO study meta-data.

• Negative controls: Knockdown/knockout if available; or tissue with minimal expression (guided by PubMed evidence).

• Storage: −20 °C to −80 °C long-term; 4 °C short-term (≤1–2 weeks).

• Ship conditions: Ice packs or ambient (stabilized), per label.

• Safety & handling: Lab PPE; follow institutional biosafety procedures.

Implementation tips for higher-quality figures & analysis

• Normalize signal to total protein or housekeeping markers; document membrane stain and normalization pipeline; quantify bands in ImageJ with linear range checks.

• For IHC, report retrieval buffer, pH, temperature, and time; include both chromogenic and fluorescence images; annotate scale bars.

• For IF/ICC, standardize laser power and gain; provide single-channel and merged panels; save raw 16-bit TIFFs.

• Archive raw data and protocols; align reports to NIH Rigor.

FAQ (indexable, non-YMYL)

Q: How was the immunogen chosen?
A: A peptide/fragment from CST11 with minimal homology to related cystatins, using CDD and BLAST screens to reduce off-target binding.

Q: What if multiple bands appear?
A: Confirm isoform sizes (NCBI Protein), assess sample processing, and include peptide competition and knockdown controls.

Q: How to design orthogonal qPCR primers?
A: Use Primer-BLAST against RefSeq/CCDS to avoid pseudogenes and non-target transcripts.

Q: Where to verify genomic coordinates?
A: UCSC Genome Browser with hgPcr for amplicon projections.