CNBP Antibody
Description
Introduction to CNBP Antibodies
CNBP (Cellular Nucleic Acid-Binding Protein) antibodies are immunological reagents designed to detect and study CNBP, a 19-kDa protein with seven zinc-finger domains involved in transcriptional regulation, translational control, and immune responses . These antibodies are critical for investigating CNBP's roles in diseases like myotonic dystrophy type 2 (DM2), viral infections, and immune dysregulation .
Applications in Research
CNBP antibodies are utilized across diverse experimental workflows:
-
Western Blot (WB): Detects CNBP at ~19–25 kDa in human brain, primary myoblasts, and cell lines (e.g., A549-ACE2, U-251 MG) .
-
Immunofluorescence (IF): Localizes CNBP in nuclei and cytoplasm, revealing its translocation during immune activation .
-
Immunoprecipitation (IP): Isolates CNBP-protein complexes to study interactions (e.g., with c-Rel in IL-12 regulation) .
-
ELISA: Quantifies CNBP expression in pathological contexts like DM2 or viral infections .
Validation and Specificity
CNBP antibodies are validated using:
-
Immunogen Specificity: Peptides or recombinant proteins from the N-terminal region (e.g., residues 1–100 in ab272676) .
-
Positive Controls: Human brain lysates, 293T cells, or SH-SY5Y neuroblastoma cells .
-
Cross-Reactivity: Most antibodies target human CNBP, with some reactive to mouse, rat, and dog .
Example Validation Data:
-
ab272676 (Abcam): Detects CNBP in RT4 bladder cancer cells and human liver tissue .
-
14717-1-AP (Proteintech): Confirmed specificity in human primary myoblasts .
Immune Regulation
-
IL-12 and Th1 Immunity: CNBP antibodies identified CNBP as a transcriptional regulator of c-Rel, essential for IL-12β production in macrophages and dendritic cells during Toxoplasma gondii infection .
-
Sustained Inflammation: CNBP knockdown via siRNA reduced LPS-induced IL-6 expression, validated by Western blot .
Antiviral Defense
-
SARS-CoV-2 Restriction: CNBP overexpression reduced viral replication by disrupting RNA-protein condensates, demonstrated using anti-CNBP antibodies in A549-ACE2 cells .
-
Type I Interferon Induction: CNBP-deficient mice showed impaired IFN production during RNA virus infections .
Immunogen Sequences
Product Specs
Q&A
What are the primary applications of CNBP antibodies in molecular biology?
CNBP antibodies are used to study the protein’s localization, expression dynamics, and interactions with nucleic acids. Key applications include:
-
Western blot (WB): Detect CNBP isoforms (19.5 kDa) in lysates from model systems (e.g., human, mouse, zebrafish) .
-
Immunofluorescence (IF): Visualize CNBP’s nuclear-cytosolic translocation under stimuli like lipopolysaccharide (LPS) .
-
Chromatin immunoprecipitation (ChIP): Map CNBP-DNA interactions at promoters of sustained immune genes (e.g., IL-6) .
-
RNA immunoprecipitation (RIP): Identify CNBP-RNA complexes, such as its stabilization of CCND1 mRNA via 5’UTR binding .
Methodological Tip: Validate antibody specificity using cnbp-knockout cell lines. For example, LPS-treated macrophages show reduced nuclear CNBP signal upon siRNA knockdown, confirming target specificity .
How should researchers validate CNBP antibody specificity?
Three orthogonal validation strategies are recommended:
-
Genetic Knockdown: Compare signal intensity in wild-type vs. cnbp CRISPR/Cas9-KO cells (WB/IF) .
-
Competitive Peptide Blocking: Pre-incubate antibody with recombinant CNBP (1–170 aa). A >80% reduction in signal confirms specificity .
-
Cross-Species Reactivity: Test antibody performance in phylogenetically distant species (e.g., zebrafish), as CNBP’s N-terminal domain is highly conserved .
Common Pitfall: Commercial antibodies may cross-react with paralogs like ZNF9. Always verify using KO controls .
What factors influence CNBP antibody performance in immunohistochemistry (IHC)?
CNBP’s phosphorylation-dependent dimerization alters epitope accessibility. Optimize protocols by:
-
Fixation: Use 4% PFA (10 min) to preserve dimerized CNBP conformations .
-
Antigen Retrieval: Citrate buffer (pH 6.0, 95°C, 20 min) improves epitope exposure in FFPE tissues .
-
Blocking: 5% BSA + 0.1% Tween-20 reduces non-specific binding to G-rich nucleic acid regions .
Data Interpretation Note: Nuclear CNBP localization post-LPS treatment correlates with NF-κB activation (r = 0.78, p < 0.01) .
How does CNBP regulate sustained cytokine expression in innate immunity?
CNBP maintains IL-6 transcription via a dual mechanism:
-
DNA Binding: CNBP recognizes G-rich motifs (e.g., 5’-GGAG-3’) in the IL-6 promoter, confirmed by EMSA and DNA pull-down assays .
-
Autoregulatory Loop: LPS induces NF-κB-mediated cnbp transcription, creating a feedforward loop that prolongs cytokine production .
Experimental Design:
-
Time-Course Analysis: Measure CNBP nuclear accumulation (IF) and IL-6 mRNA (qPCR) at 0–24 hr post-LPS.
-
Pharmacological Inhibition: Treat cells with BAY11-7082 (NF-κB inhibitor) to disrupt CNBP induction .
What experimental approaches resolve contradictions in CNBP’s role in RNA stability?
Discrepancies arise from cell type-specific RNA binding partners. To address this:
-
RIP-Seq: Identify CNBP-associated RNAs in your model system. In colorectal cancer cells, CNBP stabilizes CCND1 mRNA via LAST lncRNA bridging .
-
Domain Mapping: Truncate CNBP’s zinc finger domains (ΔZF1-7) to assess RNA binding dependency .
Key Data:
| Condition | CCND1 mRNA Half-Life | CNBP Localization |
|---|---|---|
| Wild-Type | 9.0 ± 0.5 hr | Nuclear/Cytosol |
| CNBP-KD | 3.2 ± 0.3 hr* | Cytosolic |
| *Data from HCT116 cells |
How can researchers study CNBP-RNA interactions in live cells?
Use biotinylated RNA pull-down assays with the following steps:
-
Probe Design: Synthesize biotinylated RNAs containing G-rich sequences (e.g., CCND1 5’UTR) .
-
Streptavidin Capture: Incubate lysates with probes, isolate complexes, and detect CNBP via WB .
-
EMSA Validation: Confirm direct binding using purified CNBP and Cy3-labeled RNA (K<sub>d</sub> = 12 nM) .
Troubleshooting: Include scramble RNA controls to rule out non-specific interactions.
What in vivo models are suitable for studying CNBP’s immune functions?
Zebrafish (Danio rerio):
-
Advantages: Conserved NF-κB pathway, transparent embryos for live imaging .
-
Protocol: Inject cnbp morpholinos into embryos, infect with Shigella flexneri, and monitor survival (≤40% in morphants vs. 85% in controls) .
Murine Macrophages:
Product Science Overview
Cellular Nucleic Acid Binding Protein (CNBP), also known as zinc finger protein 9, is a highly conserved protein that plays a crucial role in various biological processes. It is known for its involvement in transcription regulation, cell proliferation, and apoptosis during vertebrate organogenesis . CNBP is characterized by its seven tandem cysteine-cysteine-histidine-cysteine (CCHC) zinc-knuckle repeats and a glycine/arginine-rich region, which is similar to the arginine-glycine-glycine (RGG) box of RNA-binding proteins .
CNBP acts as a nucleic acid chaperone, regulating the transcription of several genes, including c-myc, wnt, and skeletal muscle chloride channel 1 (clc1) . It also inhibits the translation of ribosomal protein mRNAs (rp-mRNAs) . CNBP has been implicated in various human diseases, such as myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) .
CNBP is a key transcriptional regulator required for activating the innate immune response. It binds to specific motifs in the promoter region of inflammatory cytokines, directly inducing the expression of target genes . For example, lipopolysaccharide (LPS) induces CNBP expression through an NF-κB-dependent manner, enabling prolonged interleukin-6 (IL-6) gene expression . This process is crucial for maintaining the immune response, as demonstrated by the increased susceptibility of CNBP-depleted zebrafish to Shigella flexneri infection .
The mouse anti-human CNBP antibody is a monoclonal antibody used in various research applications to study the function and regulation of CNBP in human cells. This antibody is designed to specifically bind to human CNBP, allowing researchers to investigate its role in different cellular processes and disease states.
The mouse anti-human CNBP antibody is widely used in immunohistochemistry, Western blotting, and immunoprecipitation assays to detect and quantify CNBP in human tissues and cell lines. It is also employed in studies exploring the molecular mechanisms underlying CNBP-mediated transcription regulation, immune response, and disease pathogenesis.
