RIBC2 Antibody

What is RIBC2 and why is it significant in research?

RIBC2 (RIB43A domain with coiled-coils 2, also known as C22orf11) is a protein that has garnered research interest due to its potential roles in cellular functions. Current evidence suggests it may have tissue-specific expression patterns with notable presence in brain tissue, testis, and some cancer tissues . Research significance includes:

• RIBC2 has been observed to have altered expression in several cancer types, including upregulation in breast cancer and downregulation in ovarian and kidney renal clear cell carcinoma

• The protein contains RIB43A domains and coiled-coil regions that suggest potential structural or protein interaction functions

• Genetic variants like rs2272804 in the 5'UTR of RIBC2 significantly affect its expression levels, making it an interesting model for studying genetic regulation mechanisms

What applications are RIBC2 antibodies commonly used for?

RIBC2 antibodies have been validated for multiple experimental applications:

• Western Blot (WB): Typically used at 1:500-1:5000 dilution, with observed molecular weight of approximately 37kDa in human brain tissue

• Immunohistochemistry (IHC): Used at 1:20-1:200 dilution for paraffin-embedded tissues with recommended HIER pH 6 retrieval

• Immunofluorescence (IF): Applied at 1:10-1:100 dilution, validated in cell lines such as HeLa

• ELISA: Various commercial antibodies are validated for ELISA applications

What is the typical species reactivity profile of RIBC2 antibodies?

Most commercially available RIBC2 antibodies demonstrate the following reactivity profile:

• Human: Strong reactivity confirmed in multiple tissues including brain, colon cancer samples, and cell lines

• Mouse: Reactivity reported but typically with less extensive validation

• Rat: Reactivity reported but typically with less extensive validation

When selecting an antibody for cross-species applications, validation data specific to your target species should be carefully reviewed, as reactivity can vary significantly between antibody clones.

What are the optimal sample preparation protocols for RIBC2 antibody applications?

For Western Blot:

• Tissue extraction should be performed in standard RIPA buffer with protease inhibitors

• Brain tissue samples have demonstrated good RIBC2 detection with standard SDS-PAGE protocols

• Recommended protein loading: 20-40μg of total protein per lane

• Transfer conditions: Standard wet transfer to PVDF membranes (100V for 60-90 minutes)

For Immunohistochemistry:

• Fixation: 10% neutral buffered formalin, 24 hours at room temperature

• HIER (Heat-Induced Epitope Retrieval) at pH 6 is strongly recommended

• Blocking: 5-10% normal serum (species different from primary antibody source) in PBS with 0.1% Triton X-100

• Primary antibody incubation: Overnight at 4°C at 1:200-1:500 dilution

For Immunofluorescence:

• Fixation: 4% paraformaldehyde in PBS, 15 minutes at room temperature

• Permeabilization: 0.1-0.5% Triton X-100 in PBS, 10 minutes

• Blocking: 1% BSA, 10% normal serum in PBS, 1 hour at room temperature

• Primary antibody incubation at 1:10-1:100 dilution

How can I validate the specificity of a RIBC2 antibody?

A multi-approach validation strategy is recommended:

• Positive Control Tissues: Human brain tissue and colon cancer samples have demonstrated reliable RIBC2 expression

• Recombinant Protein Controls:

  • Use recombinant RIBC2 protein in Western blot as positive control

  • Create a dilution series (10-100ng) to verify antibody sensitivity and linear detection range

• Knockout/Knockdown Validation:

  • siRNA knockdown of RIBC2 in expressing cell lines (e.g., HeLa cells)

  • CRISPR-Cas9 knockout cell lines when available

  • Signal reduction/elimination confirms specificity

• Peptide Blocking:

  • Pre-incubate antibody with immunizing peptide (if available)

  • Disappearance of signal in blocked sample confirms specificity

• Molecular Weight Verification:

  • Expected RIBC2 band at approximately 37kDa

  • Any additional bands should be thoroughly investigated

What are common troubleshooting strategies for RIBC2 Western blots?

How does the rs2272804 variant affect RIBC2 detection using antibodies?

The rs2272804 SNP in the 5'UTR of RIBC2 creates an upstream open reading frame (uORF) that significantly inhibits RIBC2 expression at both mRNA and protein levels . This has important implications for antibody-based detection:

How can RIBC2 antibodies be applied in cancer research contexts?

RIBC2 demonstrates differential expression across multiple cancer types, making it a potential subject for cancer research :

• Expression Profiling:

  • Breast cancer: RIBC2 is significantly upregulated (p < 0.0001)

  • Ovarian cancer: RIBC2 is significantly downregulated (p < 0.0001)

  • Kidney renal clear cell carcinoma: RIBC2 is significantly downregulated (p = 0.0004)

• Methodological Approach for Cancer Tissue Studies:

  • Use tissue microarrays (TMAs) for high-throughput screening

  • Paired tumor/normal samples are essential for comparative analysis

  • Incorporate clinical data correlation (stage, grade, survival)

  • Consider genetic background (rs2272804 genotype) in expression analysis

• Co-expression Studies:

  • RIBC2 may interact with cancer-relevant proteins like TRAF2 and TRIM37

  • Co-immunoprecipitation protocols:

    • Crosslink with 1-2% formaldehyde for 10 minutes

    • Sonication on ice to shear chromatin

    • Immunoprecipitate with RIBC2 antibody

    • Western blot for interacting partners

• Functional Studies in Cancer Cell Lines:

  • Combine RIBC2 antibody-based detection with manipulation of RIBC2 expression

  • Correlate RIBC2 levels with cellular phenotypes and cancer-related pathways

What are the technical considerations for using RIBC2 antibodies in chromatin immunoprecipitation (ChIP) assays?

While RIBC2 itself is not a transcription factor, research suggests it may interact with transcriptional regulators like TRIM37 . For ChIP applications:

• Crosslinking and Chromatin Preparation:

  • Formaldehyde treatment: 37% formaldehyde for 10 minutes at room temperature

  • Sonication should be optimized to produce DNA fragments of 200-500bp

  • Verify fragment size by gel electrophoresis before proceeding

• Immunoprecipitation:

  • Use 2-5μg of RIBC2 antibody per reaction

  • Include appropriate controls:

    • IgG from same species as negative control

    • Known chromatin-associated protein antibody as positive control

    • Input samples (non-immunoprecipitated chromatin)

• PCR Amplification and Analysis:

  • Design primers for potential binding regions based on bioinformatic predictions

  • qPCR is preferred for quantitative analysis over endpoint PCR

  • Normalize to input samples and IgG control

• Data Analysis and Verification:

  • Follow up ChIP-PCR with ChIP-seq for genome-wide binding profiles

  • Analyze binding patterns in relation to histone modifications (H3K4me1, H3K4me3)

  • Verify binding sites with reporter assays

How can RIBC2 antibodies be optimized for use in immunohistochemical analysis of ciliated tissues?

Given RIBC2's potential connection to ciliated structures (as suggested by its coiled-coil domains and relation to other cilia-associated proteins), specialized protocols may be required:

• Tissue Preparation:

  • Preserve ciliary structures with gentle fixation: 4% paraformaldehyde, 6-12 hours

  • Gradual dehydration series to prevent tissue distortion

  • Careful orientation during embedding to capture ciliary structures

• Antigen Retrieval Optimization:

  • Test multiple retrieval methods:

    • Heat-induced epitope retrieval (HIER) at pH 6.0 (recommended)

    • Enzymatic retrieval with proteinase K (0.1-1μg/ml, 10-20 minutes)

    • Combination approaches for complex tissues

• Co-staining Approaches:

  • RIBC2 antibody (1:200-1:500) combined with ciliary markers:

    • Acetylated α-tubulin for axoneme

    • γ-tubulin for basal bodies

    • Sequential staining protocol to prevent antibody cross-reactivity

• Detection Systems:

  • For faint signals: Tyramide signal amplification (TSA)

  • For co-localization: Fluorescent secondary antibodies with spectral separation

  • For permanent preparations: Polymer-based detection systems

• Controls and Validation:

  • Positive control tissues with known cilia (fallopian tube shows strong membranous positivity in glandular cells)

  • Internal controls: RIBC2 expression in non-ciliated cells within the same section

What considerations should be made when using RIBC2 antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence allows simultaneous detection of multiple proteins:

• Antibody Panel Selection:

  • RIBC2 antibody compatibility with other antibodies must be verified

  • Primary antibodies should originate from different host species

  • For same-species antibodies, use directly conjugated primaries or sequential staining

• Optimized Protocol:

  • Fixation: 4% paraformaldehyde, 15-20 minutes

  • Permeabilization: 0.2% Triton X-100, 10 minutes

  • Blocking: 10% normal serum + 1% BSA, 1 hour

  • Primary antibody cocktail: RIBC2 (1:50) with other antibodies

  • Secondary antibody selection: Minimal cross-reactivity formulations

• Spectral Considerations:

  • RIBC2 can be visualized with various fluorophores (Alexa Fluor 488, 555, 647)

  • FITC-conjugated RIBC2 antibodies are commercially available

  • Plan fluorophore combinations to minimize spectral overlap

  • Include single-stain controls for spectral unmixing

• Analysis Methods:

  • Confocal microscopy for high-resolution co-localization studies

  • Quantitative image analysis:

    • Colocalization coefficients (Pearson's, Mander's)

    • Intensity correlation analysis

    • Distance measurement between structures

How can I design experiments to investigate the relationship between RIBC2 genetic variants and protein expression?

The rs2272804 variant significantly affects RIBC2 expression . To investigate:

• Genotype-Phenotype Correlation Study:

  • Genotype samples for rs2272804 (C/C, C/A, A/A)

  • Quantify RIBC2 protein using calibrated Western blot

  • Statistical analysis of expression levels across genotype groups

• Functional Validation using Reporter Assays:

  • Clone wild-type (C) and variant (A) 5'UTR sequences upstream of luciferase

  • Transfect constructs into relevant cell lines

  • Measure luciferase activity as a proxy for translational efficiency

  • Normalization controls: co-transfected Renilla luciferase

• mRNA and Protein Correlation Analysis:

  • RT-qPCR for mRNA quantification

  • Western blot with RIBC2 antibody for protein quantification

  • Calculate protein:mRNA ratio to assess translational efficiency

  • Compare ratios across genotype groups

• Tissue-Specific Effects Investigation:

  • Utilize tissue samples of known genotype

  • Immunohistochemistry with calibrated conditions

  • Comparative digital pathology quantification

  • Account for tissue-specific regulatory factors

How can RIBC2 antibody data be integrated with other -omics data for systems biology approaches?

Integration of antibody-based data with other -omics approaches:

• Multi-omics Experimental Design:

  • Parallel analysis of the same samples using:

    • RIBC2 antibody-based proteomics

    • Transcriptomics (RNA-seq)

    • Genomics (rs2272804 genotyping)

    • Interactomics (co-IP followed by mass spectrometry)

• Data Integration Methods:

  • Correlation analysis between RIBC2 protein levels and:

    • RIBC2 mRNA expression

    • Expression of interacting partners (TRAF2, TRIM37, VIM, UBASH3B)

    • Pathway activation markers

• Network Analysis:

  • Position RIBC2 within protein-protein interaction networks

  • Utilize STRING database predictions as starting points

  • Validate key interactions with co-IP using RIBC2 antibodies

  • Map genetic variants to network perturbations

• Visualization and Analysis Tools:

  • Use tools like Cytoscape for network visualization

  • Apply machine learning approaches to identify patterns

  • Develop predictive models incorporating RIBC2 expression

What are the methodological considerations for studying RIBC2 in the context of cancer biomarker research?

Given RIBC2's differential expression in multiple cancers :

• Biomarker Validation Pipeline:

  • Discovery Phase:

    • IHC screening across cancer tissue microarrays

    • Quantitative assessment using digital pathology

    • Statistical comparison with clinical parameters

  • Validation Phase:

    • Independent cohort testing with standardized protocols

    • Multivariate analysis with established biomarkers

    • Survival analysis stratified by RIBC2 expression

  • Clinical Implementation Considerations:

    • Standardized staining protocols

    • Scoring system development

    • Quality control measures

• Technical Standardization:

  • Antibody validation using multiple approaches

  • Automated staining platforms for reproducibility

  • Standard positive controls for inter-laboratory comparison

• Complementary Approaches:

  • Combine RIBC2 IHC with DNA/RNA-based rs2272804 testing

  • Multiplex IHC panels including RIBC2 and interacting proteins

  • Liquid biopsy validation (if applicable)

• Reporting Standards:

  • Follow REMARK guidelines for biomarker studies

  • Document all antibody validation steps

  • Include genotype information when available