CYP26B1 Polyclonal Antibody — Technical Research Guide (RUO)
Scientific background (research context only)
CYP26B1 is a microsomal cytochrome P450 monooxygenase that catalyzes oxidative catabolism of all-trans-retinoic acid (atRA), shaping retinoid gradients during development and in adult tissues. Core reference resources include the NCBI Gene record for CYP26B1 (human) for locus, curated aliases, and RefSeqs (see NCBI Gene). Protein-level curation, including conserved domains and post-translational features, is accessible via NCBI Protein (NCBI Protein). Retinoid chemistry and pathways are covered by PubChem entries for atRA and hydroxylated products (PubChem compound overview). Canonical, peer-reviewed pathway chapters are available through NCBI Bookshelf (e.g., retinoid signaling basics: NCBI Bookshelf).
For genome context (coordinates, intron–exon structure, orthology tracks), consult the UCSC Genome Browser on hg38 or appropriate assemblies (UCSC Browser). Expression matrices and perturbation datasets can be surveyed in GEO (GEO portal) and raw sequencing runs in SRA (SRA). Curated coding sequence sets are listed in CCDS (CCDS project). For sequence identifiers used in primer or peptide design, use RefSeq transcripts and proteins (RefSeq overview).
Additional background reading and standards
• Assay performance reporting and measurement science guidance: NIST reference resources (NIST portal).
• FAIR data deposition standards and bioinformatics pipelines: NIH Data Sharing hub (NIH sharing).
• Chemical safety data and RA handling: NIH OHSRP general resources (NIH OHSRP).
Antigen design and antibody characteristics
A typical polyclonal against CYP26B1 is raised to a peptide(s) unique to CYP26B1 (avoiding homology with CYP26A1/CYP26C1). When selecting or validating an antibody, verify that the immunogen sequence maps to an isoform-invariant region and is low-complexity filtered (consult conserved-domain and BLAST reports: Conserved Domain at NCBI, BLAST). Predicted topology, signal peptides, and ER-targeting features are also viewable via NCBI Protein feature tables (feature tables).
Species reactivity (in-silico guidance): Align immunogen to orthologs downloaded from NCBI Gene or HomoloGene (archival) and view alignments in UCSC multiple alignment tracks (UCSC multiple alignments). This helps forecast cross-reactivity for mouse/rat/primate studies (lab validation still required).
Recommended research applications (RUO)
The following are research-use protocols only. Always confirm with local SOPs and institutional guidance.
Western Blot (WB)
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Sample: ER-enriched lysates or total protein from RA-responsive cell lines.
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Lysis: RIPA with protease inhibitors (consult institutional protocol libraries, e.g., UCLA MIMG resources).
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Load: 20–40 µg per lane; include a positive control lysate (RA-treated cells) and negative control (siRNA knockdown).
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Electrophoresis/transfer: Standard SDS-PAGE to PVDF; transfer QC per university core guidance (e.g., Iowa State lab resources).
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Primary incubation: Optimize 1:500–1:2000; validate linear range.
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Specificity checks: Peptide competition (if immunogen available) and knockdown/overexpression comparisons.
General WB troubleshooting and gel chemistry primers can be found at NIH-hosted teaching resources (NIH OITE training).
Immunohistochemistry—Paraffin (IHC-P)
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Fixation: Neutral buffered formalin; antigen retrieval (citrate pH 6 or EDTA pH 9) titrated per tissue.
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Controls: Adjacent section w/o primary; peptide competition where feasible.
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Interpretation: ER-localized signals; compare across tissues with known RA gradients. For image analysis fundamentals, see NLM resources on digital imaging (NLM).
Immunofluorescence (IF)
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Cells: RA-responsive lines; pre-treat with atRA (consult PubChem handling notes: PubChem retinoic acid).
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Permeabilization: 0.1–0.3% Triton X-100; co-stain ER markers.
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Microscopy: Exposure kept within linear dynamic range; see Colorado microscopy educational pages for basic guidance (UC Boulder MCDB).
Immunoprecipitation (IP)
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Beads: Protein A/G; 2–5 µg antibody per 1 mg lysate.
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Readout: WB against CYP26B1 or LC-MS/MS peptide ID. For MS-ready prep, reference NIH Core best practices (NIH core facilities).
Positive/negative controls and perturbations
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Chemical: atRA dosing and time-course to modulate CYP26B1 levels (PubChem atRA).
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Genetic: siRNA/CRISPR knockdown for loss-of-signal; overexpression of tagged CYP26B1 for band-position confirmation (plasmid sequence verification via NCBI Nucleotide: Nucleotide).
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Tissue panels: Use expression atlases and datasets discoverable through GEO to choose RA-responsive tissues (GEO portal).
Data management, identifiers, and reproducibility
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Primary identifiers: Gene (e.g., CYP26B1), RefSeq mRNA/protein accessions (see RefSeq), CCDS IDs (CCDS).
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Genome coordinates: Confirm on UCSC (UCSC Browser).
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Reagent reporting: Follow measurement-science guidance from NIST for documentation of antibody lots, dilutions, membranes, and blocking buffers (NIST resources).
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Data deposition: Upload raw/processed results to GEO or SRA with complete methods (GEO, SRA).
Troubleshooting matrix (condensed)
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Weak/absent band in WB → Increase lysate load; verify transfer; extend primary incubation; confirm RA induction; cross-check antibody stability (see general troubleshooting at NIH training: NIH OITE).
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Multiple bands → Titrate antibody; adjust blocking; include peptide competition; verify isoform/processing predictions via NCBI Protein feature maps (NCBI Protein).
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High background in IHC/IF → Optimize antigen retrieval; shorten DAB development; raise stringency washes; validate secondary only. Image handling guidance at NLM (NLM).
Storage, shipping, and handling (general RUO guidance)
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Storage: Aliquot upon receipt; avoid freeze–thaw; follow standard bioreagent practices from institutional biosafety offices (e.g., NIH resources: NIH biosafety).
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Working dilution stability: Keep on ice during setup; discard mixed working solutions after use per local SOPs.
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Safety: Consult SDS for buffers and azide if present; chemical safety primers at NIH (NIH OHSRP).
Example method outline (WB) for CYP26B1 detection
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Culture RA-responsive cells; treat with 0.1–1 µM atRA for 6–24 h (PubChem atRA).
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Lyse in RIPA + inhibitors; quantify protein (BCA).
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Resolve 30 µg on 8–12% SDS-PAGE; transfer to PVDF.
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Block 5% BSA; incubate CYP26B1 pAb 1:1000 overnight 4 °C.
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Wash; HRP secondary 1:5000; ECL; image in linear range.
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Validate specificity by siRNA knockdown (sequence design checked via BLAST: BLAST).
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Report identifiers (Gene, RefSeq, CCDS) and full buffer compositions; see NIH data-sharing expectations (NIH sharing).
FAQ (research-centric)
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Q: How do I confirm band identity?
A: Use knockdown/overexpression, peptide competition, and migration versus predicted MW from NCBI Protein (Protein). -
Q: Which exon does my immunogen map to?
A: Load the peptide as a custom track on UCSC and intersect with exon annotation (UCSC custom tracks). -
Q: Where can I find public CYP26B1 expression datasets?
A: Search GEO for “CYP26B1” in RNA-seq or microarray series (GEO search).
SEO additions for your product page
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Suggested H2s: “CYP26B1 Antibody for Retinoic-Acid Catabolism (RUO)”, “Validated for WB, IHC-P, IF, and IP”, “Retinoid Gradient Biology—Research Tools”
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Image alt-text examples: “CYP26B1 polyclonal antibody western blot in RA-treated cells”, “CYP26B1 IHC staining ER-enriched signal (research-use)”.
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Internal linking ideas: Link to other RUO retinoid-pathway antibodies (e.g., CYP26A1, RAR/RXR) and RA pathway reagents (reference at NCBI Bookshelf Bookshelf).
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Structured data (summary): Use Product schema with
isAccessoryOrSparePartForreferencing retinoid research kits; keep claims strictly research-use and avoid clinical language.
Compliance note (place near footer)
This CYP26B1 antibody is For Research Use Only (RUO) and not for use in diagnostic procedures. Follow institutional biosafety and chemical-safety guidance (see NIH resources: NIH biosafety).