Cirbp Antibody

What is CIRBP and why is it important in research?

CIRBP is a cold-inducible RNA-binding protein with a calculated molecular weight of 19 kDa (172 amino acids) that functions as both a nuclear protein and a stress response mediator. It plays crucial roles in regulating mRNA stability and translation of target genes involved in cell survival, proliferation, and stress responses. CIRBP has gained significant research interest due to its dual role in cancer biology, functioning both as an oncogene and tumor suppressor depending on the cellular context . Researchers study CIRBP to understand stress response mechanisms, inflammation pathways, and cancer progression.

What applications are validated for CIRBP antibodies?

CIRBP antibodies have been validated for multiple applications with specific dilution recommendations:

These applications have been documented in numerous publications, with WB featuring in 67 publications, IHC in 14 publications, and IF in 12 publications according to recent antibody validation data .

Which cell lines and tissues show positive reactivity with CIRBP antibodies?

Validated western blot detection has been confirmed in:

• Cell lines: Y79, A549, HEK-293, HepG2, PC-12, PC-13

• Tissues: Mouse testis tissue, rat testis tissue

• IHC positivity: Human pancreas cancer tissue, human breast cancer tissue, mouse pancreas tissue

The antibody shows cross-reactivity with human, mouse, rat, and squirrel samples, making it versatile for comparative studies across species .

What is the optimal protocol for CIRBP immunohistochemistry in tissue samples?

For optimal CIRBP detection in tissue samples:

• Tissue preparation: Formalin-fixed, paraffin-embedded sections at 4-6 μm thickness

• Antigen retrieval: Primary recommendation is TE buffer (pH 9.0), with citrate buffer (pH 6.0) as an alternative

• Blocking: Use 1% donkey serum in PBS/0.1% Tween-20 for 10 minutes at room temperature

• Primary antibody: Apply CIRBP antibody at 1:50-1:500 dilution for 45-60 minutes at room temperature

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody for 30 minutes

• Visualization: Use DAB or other chromogen, with hematoxylin counterstain

• Scoring: Implement the Fromowitz standard based on staining intensity (0-3) and proportion of positive cells (0-4) to calculate an H-score

This protocol has been validated in pancreatic cancer tissue microarray studies where H-scores ≥3 defined CIRBP-high expression groups for clinical correlation studies .

How should researchers quantify CIRBP expression in imaging studies?

For quantitative measurement of CIRBP expression in imaging studies:

• Image acquisition: Use confocal microscopy with identical settings for all samples within an experiment

• ROI selection: For stress granule association studies, identify regions of interest using the wand tool in ImageJ/Fiji based on G3BP1 (stress granule marker) staining

• Fluorescence measurement: Determine mean fluorescence intensity in the EGFP channel (for CIRBP-EGFP fusion proteins)

• Enrichment calculation: To determine CIRBP enrichment, compare the ROI of G3BP1-positive cytoplasmic condensates to a 0.98-pixel band around the condensate (representing cytoplasmic intensity)

• Background correction: Apply background correction to all values

• Statistical analysis: Perform in GraphPad Prism using appropriate statistical tests

This methodology was validated in a study examining CIRBP phosphorylation and methylation, where at least 10 cells and 44 stress granules were analyzed per condition .

What controls should be included when using CIRBP antibodies in experimental procedures?

Essential controls for CIRBP antibody experiments:

• Positive controls:

  • Known CIRBP-expressing cells (HepG2, A549, SW1990 for high expression)

  • Cold-treated samples (4°C exposure induces CIRBP expression)

• Negative controls:

  • CIRBP knockdown/knockout samples using validated shRNA sequences:

    • CIRBP sh#1: 5′-CATGAATGGGAAGTCTGTA-3′

    • CIRBP sh#2: 5′-TCTCAAAGTACGGACAGAT-3′

  • Non-targeting control: 5′-TTCTCCGAACGTGTCACGT-3′

• Loading controls for western blots:

  • β-actin or GAPDH antibodies for protein normalization

• Antibody validation:

  • Peptide competition assays

  • Multiple antibody comparison (using different epitope targets)

These controls have been validated in multiple published studies and ensure experimental rigor when investigating CIRBP function .

How do post-translational modifications affect CIRBP antibody recognition?

CIRBP undergoes several post-translational modifications that can impact antibody recognition:

• Phosphorylation: The RG/RGG region of CIRBP is phosphorylated by serine-arginine protein kinase-1 (SRPK1), which can mask epitopes in this region. Researchers should be aware that phosphorylation status may affect antibody binding, particularly for antibodies targeting the C-terminal region .

• Arginine methylation: CIRBP is also subject to arginine methylation, which regulates its interaction with transportin-1 and cellular localization. This modification can alter antibody accessibility to certain epitopes .

• Recommendations for analysis:

  • Use phosphorylation-specific antibodies when studying CIRBP regulation

  • Consider using both N-terminal and C-terminal targeting antibodies to ensure detection regardless of modification state

  • Include phosphatase treatment controls when necessary to distinguish modification-dependent epitope masking

These modifications are particularly important when studying CIRBP's stress response functions, as phosphorylation regulates CIRBP's role in stress granule association .

How can CIRBP antibodies help elucidate its dual role in cancer biology?

CIRBP exhibits context-dependent roles in cancer that can be investigated using antibodies:

• Tumor-promoting functions:

  • Use CIRBP antibodies to examine nuclear vs. cytoplasmic localization in IHC/IF studies

  • Correlate expression with prognostic markers

  • Investigate co-localization with oncogenic partners

• Tumor-suppressive functions:

  • Analyze CIRBP expression in relation to p53 binding and regulation

  • Study ferroptosis induction pathways

  • Examine cold-induction effects on cancer cell viability

• Methodological approach:

  • Perform subcellular fractionation followed by western blotting

  • Use tissue microarrays with survival data correlation

  • Combine with RNA immunoprecipitation (RIP) to identify bound mRNAs

What is the optimal approach for RNA immunoprecipitation (RIP) using CIRBP antibodies?

For RNA immunoprecipitation to study CIRBP-RNA interactions:

• Cell lysis: Prepare lysate in a buffer containing protease inhibitors (e.g., 10 mM Tris-HCl pH 7.5, 120 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) with RNase inhibitors

• Pre-clearing: Incubate lysate with protein A/G beads for 1 hour at 4°C

• Immunoprecipitation: Add CIRBP antibody (typically 2-5 μg) to pre-cleared lysate and incubate overnight at 4°C

• Bead capture: Add protein A/G beads and incubate for 2-3 hours at 4°C

• Washing: Perform stringent washes to remove non-specific interactions

• RNA extraction: Isolate bound RNA using TRIzol or equivalent reagent

• Analysis: Perform RT-qPCR or RNA-seq to identify bound transcripts

This protocol successfully demonstrated direct binding of CIRBP to p53 RNA in pancreatic cancer cells, providing evidence for CIRBP's role in regulating ferroptosis through the p53/GPX4 pathway .

What are common issues with CIRBP western blotting and how can they be resolved?

Common western blot problems and solutions:

• Multiple bands:

  • Issue: CIRBP has a calculated molecular weight of 19 kDa, but additional bands may appear

  • Solution: Include CIRBP knockdown controls; use reducing agents to eliminate potential aggregates; test different antibody concentrations (1:1000-1:4000)

• Weak signal:

  • Issue: Low endogenous CIRBP expression in some cell types

  • Solution: Cold-treat cells (32°C or lower) for 24 hours to induce CIRBP expression; increase protein loading to 50-80 μg; optimize blocking conditions and antibody concentrations

• High background:

  • Issue: Non-specific binding or inadequate blocking

  • Solution: Use 5% BSA instead of milk for blocking; increase washing steps; titrate antibody concentration; include 0.1% Tween-20 in washing buffer

• Species-specific issues:

  • Issue: Different detection efficiency across species

  • Solution: Verify antibody reactivity (human, mouse, rat confirmed); adjust antibody concentration for cross-species work

How can researchers validate knockdown/knockout models for CIRBP antibody specificity?

For validating CIRBP knockdown/knockout models:

• shRNA validation:

  • Use multiple shRNA constructs targeting different CIRBP regions

  • Recommended sequences: CIRBP sh#1 (5′-CATGAATGGGAAGTCTGTA-3′) and CIRBP sh#2 (5′-TCTCAAAGTACGGACAGAT-3′)

  • Include non-targeting control: 5′-TTCTCCGAACGTGTCACGT-3′

• Protein verification:

  • Perform western blot with CIRBP antibody

  • Quantify knockdown efficiency using densitometry (Quantity One 4.62 or similar software)

  • Aim for >80% reduction in protein levels

• Functional validation:

  • Confirm altered expression of CIRBP-regulated genes (p53, DPP4, NOX1, FTH1, GPX4)

  • Evaluate phenotypic changes (proliferation, apoptosis, stress response)

  • Test cold induction response in knockdown models

• In vivo validation:

  • Subcutaneous injection of knockdown cells in mouse models

  • Perform IHC on tumor tissues with CIRBP antibody

  • Compare tumor growth parameters with control groups

These approaches were successfully employed in pancreatic cancer studies where CIRBP knockdown models were used to demonstrate its role in tumor development and chemosensitivity .

How does CIRBP influence stress granule formation and what methods can assess this?

CIRBP's role in stress granule (SG) formation can be studied using specialized techniques:

• Semi-permeabilized cell systems:

  • Prepare cells with digitonin permeabilization to selectively permeabilize the plasma membrane

  • Apply recombinant CIRBP-EGFP to cells

  • Wash cells (3 x 5 min in KPB buffer on ice) to remove unbound proteins

  • Perform immunofluorescence for G3BP1 (SG marker) using primary antibody (rabbit anti-G3BP1, 1:200-1:500)

  • Apply secondary antibody (Alexa 555 Donkey-anti-Rabbit, 1:500)

  • Stain DNA with DAPI (0.5 mg/mL)

  • Mount in antifade mounting medium

  • Analyze by confocal microscopy

• Quantification approach:

  • Use ImageJ/Fiji for image analysis

  • Identify SGs as ROIs using G3BP1 staining

  • Measure mean fluorescence intensity of CIRBP-EGFP within SGs

  • Compare different CIRBP constructs (wild-type vs. phosphomutants)

  • Analyze ≥10 cells and ≥44 SGs per condition

This methodology revealed that phosphorylation of CIRBP regulates its association with stress granules, providing insights into its function during cellular stress responses .

What is the relationship between CIRBP and ferroptosis in cancer, and how can antibodies help investigate this?

The CIRBP-ferroptosis relationship in cancer can be investigated using these approaches:

• Protein expression analysis:

  • Western blot for CIRBP and ferroptosis markers (p53, GPX4, DPP4, NOX1, FTH1)

  • Cold treatment (32°C or lower) to induce CIRBP expression

  • Compare expression levels between normal and cancer tissues/cells

• Functional assays:

  • Fe2+ accumulation measurement using iron probes

  • ROS generation assessment

  • GSH-Px activity assays

  • Cell viability assays with ferroptosis inhibitors

• Mechanistic studies:

  • Co-immunoprecipitation of CIRBP with p53

  • RNA immunoprecipitation to confirm direct binding to p53 mRNA

  • Subcellular fractionation to determine CIRBP localization

  • Cold induction experiments with/without ferroptosis inhibitors

Research has demonstrated that CIRBP acts as a tumor suppressor in pancreatic cancer by inducing ferroptosis through the p53/GPX4 pathway. Cold induction significantly enhanced CIRBP expression and promoted ferroptosis by regulating key factors including p53 and GPX4, ultimately inhibiting cancer cell proliferation and inducing apoptosis .

How does CIRBP contribute to inflammatory responses and what experimental models are appropriate?

CIRBP's role in inflammation can be studied using these methodologies:

• Tissue-specific analysis:

  • Liver ischemia-reperfusion model: 70% hepatic ischemia via microvascular clamping for 60 minutes followed by reperfusion

  • Anti-CIRBP antibody treatment (1 mg/kg body weight) via jugular vein at reperfusion start

  • Blood and tissue collection 24 hours post-I/R

• Protein analysis techniques:

  • Tissue homogenization in lysis buffer (10 mM Tris-HCl pH 7.5, 120 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) with protease inhibitors

  • Sonication on ice followed by centrifugation (14,000 rpm, 15 min, 4°C)

  • Protein concentration determination (BioRad DC protein assay)

  • Western blotting for CIRBP and inflammatory markers

• Inflammatory marker assessment:

  • Measure cytokine levels (TNF-α, IL-23, IL-6, IL-1β)

  • Assess NF-κB/TLR4 signaling pathway activation

  • Quantify ROS accumulation

• In vivo models:

  • CIRBP knockout mice for comparative studies

  • Colitis-associated cancer (CAC) models

  • Survival studies following anti-CIRBP antibody treatment

Research has shown that CIRBP can induce ROS accumulation by increasing inflammatory cytokine expression, while CIRBP-knockout mice exhibited decreased inflammatory cytokine levels and attenuated ROS accumulation. Blocking CIRBP with specific antibodies provided protection against ischemia-reperfusion injury, highlighting its potential as a therapeutic target in inflammatory conditions .

How can NMR spectroscopy complement antibody-based approaches in CIRBP research?

NMR spectroscopy provides structural insights that complement antibody studies:

• Sample preparation:

  • Prepare 50-500 μM purified CIRBP protein

  • Conduct experiments at 25°C using 600-700 MHz NMR spectrometers

  • Utilize TXI or TCI triple-resonance cryoprobes

• Experimental approaches:

  • Analyze protein-RNA interactions at atomic resolution

  • Study conformational changes upon post-translational modifications

  • Investigate cold-induced structural alterations

• Integration with antibody data:

  • Validate epitope accessibility in different conformational states

  • Correlate structural changes with antibody recognition efficiency

  • Map modification sites that affect antibody binding

This combined approach provides deeper mechanistic understanding of how CIRBP structure relates to its function in stress response and RNA binding activities .

What xenograft models are most appropriate for CIRBP antibody validation in cancer research?

Validated xenograft models for CIRBP research include:

• BALB/c nude mouse model:

  • Age: 6-8 weeks old

  • Cell injection: 2 × 10^6 cells subcutaneously into the right flank

  • Experimental groups (n=5 mice/group):

    • Control (vector) + PBS

    • CIRBP-knockdown + PBS

    • Control + chemotherapy agent

    • CIRBP-knockdown + chemotherapy agent

• Measurement protocol:

  • Tumor volume calculation: V = (L × W^2)/2 (L: longest tumor axis; W: shortest tumor axis)

  • Treatment administration: Gemcitabine (80 mg/kg) or PBS intraperitoneally twice weekly for 5 weeks

  • Measurement frequency: Every 2-3 days for 40 days

• Analysis techniques:

  • Tumor excision and histological analysis

  • IHC for CIRBP expression using validated antibody dilutions

  • Western blot analysis of tumor lysates

  • Survival analysis correlation

This model successfully demonstrated that CIRBP knockdown attenuated tumor growth and enhanced chemosensitivity in pancreatic cancer, providing a robust platform for in vivo validation of CIRBP antibodies and functional studies .

How does CIRBP expression correlate with patient outcomes in cancer studies?

CIRBP expression correlation with clinical outcomes:

• Tissue microarray analysis technique:

  • Use validated CIRBP antibodies (e.g., Proteintech #10209-2-AP, 1:100-1:200 dilution)

  • Implement standardized scoring methods:

    • Staining intensity: 0 (negative), 1 (weak), 2 (moderate), 3 (strong)

    • Positive cell proportion: 0 (<1%), 1 (1-25%), 2 (26-50%), 3 (51-75%), 4 (>75%)

    • H-score calculation: multiply intensity score by proportion score (range 0-12)

  • Stratify patients into expression groups: negative (score 0), weak (1-4), moderate (5-8), strong (9-12)

  • Alternative binary classification: CIRBP-high (H-score ≥3) vs. CIRBP-low (H-score <3)

• Clinical correlation methodology:

  • Kaplan-Meier survival analysis comparing expression groups

  • Cox proportional hazards regression for multivariate analysis

  • Correlation with clinicopathological parameters

How can researchers distinguish between nuclear and cytoplasmic CIRBP localization in tissue samples?

Methods to differentiate nuclear vs. cytoplasmic CIRBP localization:

• IHC protocol optimization:

  • Fixation: 3.7% formaldehyde/PBS for 7 minutes at room temperature

  • Permeabilization: 0.5% TX-100/PBS for 5 minutes

  • Blocking: 1% donkey serum in PBS/0.1% Tween-20 for 10 minutes

  • Primary antibody: Anti-CIRBP (1:50-1:200) for 45-60 minutes

  • Counterstain: DAPI (0.5 mg/mL) for nuclear visualization

• Scoring system for subcellular localization:

  • Separate evaluation of nuclear vs. cytoplasmic staining intensity

  • Quantification of nuclear:cytoplasmic ratio

  • Digital image analysis with nuclear/cytoplasmic masking

• Validation approaches:

  • Comparison with subcellular fractionation followed by western blotting

  • Correlation with stress conditions known to affect localization

  • Cold treatment experiments (CIRBP shuttles between nucleus and cytoplasm upon cold stress)

This approach revealed that in pancreatic cancer, nuclear CIRBP expression was associated with better prognosis, while cytoplasmic levels were reduced in cancer tissues, suggesting that nuclear retention of CIRBP may be critical for its tumor-suppressive functions .

What experimental designs can elucidate the seemingly contradictory roles of CIRBP in different cancer types?

To investigate CIRBP's dual roles in cancer:

• Comprehensive expression analysis:

  • Multi-cancer tissue microarray with paired normal tissues

  • Standardized IHC protocol with CIRBP antibody (1:100-1:200)

  • Separate scoring for nuclear vs. cytoplasmic expression

  • Correlation with patient outcomes across cancer types

• Mechanistic investigations:

  • Cell-type specific knockdown and overexpression studies

  • RNA immunoprecipitation to identify cancer-type specific RNA targets

  • Analysis of post-translational modifications affecting function

  • Context-dependent protein interaction studies

• Experimental approach:

  • Compare cold-induced vs. constitutive CIRBP expression

  • Evaluate stress-dependent functions vs. homeostatic roles

  • Correlate with p53 status and ferroptosis susceptibility

  • Analyze inflammatory microenvironment effects

Research has revealed that CIRBP can function as either an oncogene or tumor suppressor depending on cancer type and cellular context. In breast cancer, CIRBP enhances oncogenic properties by downregulating CST3 mRNA, while in pancreatic cancer, it acts as a tumor suppressor by regulating p53 and inducing ferroptosis. These contradictory roles highlight the importance of context-specific analysis rather than generalizing CIRBP functions across all cancer types .