CCHCR1 Antibody, FITC conjugated

What is CCHCR1 and why is it significant in research?

CCHCR1 is a nuclear protein that has emerged as an important research target due to its multifaceted biological significance. It was first identified as a candidate gene in psoriasis and has subsequently been linked to COVID-19 susceptibility. CCHCR1 is abundantly expressed in heart, liver, skeletal muscle, kidney, and pancreas, with lower expression in lung and placenta .

The protein's significance stems from several critical aspects:

• Localization in both P-bodies and centrosomes, suggesting roles in mRNA metabolism and cell division

• Regulation of keratinocyte proliferation and differentiation, with overexpression observed in psoriatic lesions

• Sharing a bidirectional promoter with TCF19, activated by E2F1 during G1/S transition, indicating cell cycle regulatory functions

• Interaction with EDC4, a key processing body component, positioning CCHCR1 as a novel P-body component involved in mRNA metabolism

Understanding CCHCR1's functions provides critical insights into disease mechanisms related to both inflammatory conditions and potentially viral susceptibility.

How does FITC conjugation enhance CCHCR1 antibody functionality?

FITC (Fluorescein Isothiocyanate) conjugation transforms CCHCR1 antibodies into versatile visualization tools through covalent attachment of the fluorescent dye to the antibody protein structure. This chemical modification enables direct detection without secondary antibodies, significantly enhancing experimental flexibility.

The conjugation process optimally occurs under specific conditions:

• Using purified IgG (typically >95% purity, Protein G purified)

• Maintaining alkaline conditions (pH 9.5) for maximal labeling efficiency

• Reaction at room temperature for 30-60 minutes with protein concentration around 25 mg/ml

• Careful separation of optimally labeled antibodies from under/over-labeled proteins via gradient DEAE Sephadex chromatography

The resulting FITC-conjugated antibodies provide:

• Excitation at 488 nm with emission at 535 nm, compatible with standard fluorescence microscopy and flow cytometry instrumentation

• Direct visualization of CCHCR1 localization within subcellular structures

• Capability for multiplexed detection alongside other fluorophores

• Quantitative assessment of CCHCR1 expression levels in various experimental models

This modification enables precise spatial and temporal studies of CCHCR1 distribution particularly valuable for investigating its reported roles in P-bodies and centrosomes.

What are the optimal storage conditions for FITC-conjugated CCHCR1 antibodies?

Proper storage of FITC-conjugated CCHCR1 antibodies is critical for maintaining both immunoreactivity and fluorescence intensity. The following storage parameters are recommended based on manufacturer specifications:

For lyophilized preparations:

• Store at -20°C or lower in lyophilized state

• Reconstitute following manufacturer's protocol, typically using trehalose as a protectant

• After reconstitution, use immediately or prepare small aliquots for freezing

Following these guidelines ensures optimal antibody performance for up to one year after acquisition, maintaining both binding specificity and fluorescence intensity .

What applications are FITC-conjugated CCHCR1 antibodies commonly used for?

FITC-conjugated CCHCR1 antibodies serve diverse experimental applications in multiple research contexts. Their versatility stems from the combination of specific epitope recognition and direct fluorescent visualization capabilities.

The choice of epitope region significantly impacts applications, with antibodies targeting different regions (AA 281-500, AA 483-782, or AA 599-627) potentially yielding different results depending on protein conformation and interaction partners .

How do you determine the appropriate dilution ratio for FITC-conjugated CCHCR1 antibodies?

Determining optimal dilution ratios for FITC-conjugated CCHCR1 antibodies requires systematic titration to balance specific signal detection against background fluorescence. This methodological approach ensures experimental reproducibility and accurate data interpretation.

Systematic Titration Protocol:

• Begin with manufacturer recommendations:

  • IF/ICC applications: Initial range of 1:20-1:200

  • IHC applications: Initial range of 1:50-1:500

  • Flow cytometry: Typically 1:10-1:50 or approximately 2 μL per 1×10^6 cells

• Prepare serial dilutions:

  • Create 5-6 dilutions spanning the recommended range

  • Include dilutions below and above the suggested range

  • Maintain consistent diluent composition (typically blocking buffer)

• Test on appropriate controls:

  • Positive control: Human tissues/cells with confirmed CCHCR1 expression (e.g., HEK-293 cells, human colon tissue)

  • Negative control: Same sample type with primary antibody omitted

  • Isotype control: Non-specific FITC-conjugated IgG at equivalent concentration

• Evaluate signal-to-noise metrics:

  • Calculate signal-to-background ratio for each dilution

  • Assess signal intensity versus background autofluorescence

  • Consider photobleaching resistance at different concentrations

• Sample-specific optimization:

  • For tissues requiring antigen retrieval, test both TE buffer pH 9.0 and citrate buffer pH 6.0

  • Consider fixation method impacts (formaldehyde versus methanol)

  • Adjust for expression level variations across different cell/tissue types

As noted by multiple manufacturers, "It is recommended that this reagent should be titrated in each testing system to obtain optimal results" , highlighting the importance of experiment-specific optimization rather than relying solely on standard dilutions.

How can the specificity of FITC-conjugated CCHCR1 antibodies be validated for P-body localization studies?

Validating FITC-conjugated CCHCR1 antibody specificity for P-body localization studies requires comprehensive controls and experimental validation. This is particularly crucial given CCHCR1's reported interaction with EDC4, a key P-body component .

Validation Methodology:

• Co-localization with established P-body markers:

  • Perform dual immunofluorescence with FITC-CCHCR1 antibody and antibodies targeting known P-body components:

    • EDC4 (primary interaction partner of CCHCR1)

    • Decapping enzymes (DCP1/DCP2)

    • RNA-binding proteins (GW182/TNRC6)

  • Quantify co-localization using Pearson's correlation and Manders' overlap coefficients

  • Analyze distance relationships between CCHCR1 and P-body centers

• Gene silencing validation:

  • Implement siRNA or shRNA knockdown of CCHCR1

  • Compare FITC-CCHCR1 antibody signal between knockdown and control cells

  • Quantify signal reduction in P-bodies specifically

  • Include western blot validation of knockdown efficiency

• Domain-specific localization assessment:

  • Compare staining patterns using antibodies targeting different CCHCR1 regions:

    • N-terminal regions required for P-body localization

    • Other epitopes (AA 483-782 or AA 599-627)

  • Confirm expected domain-specific localization patterns

• Recombinant expression validation:

  • Generate stable cell lines expressing EGFP-tagged CCHCR1

  • Compare localization between antibody-detected endogenous CCHCR1 and EGFP-CCHCR1

  • Perform co-immunoprecipitation to confirm EDC4 interaction

• Stress response validation:

  • Apply arsenite stress to induce P-body formation

  • Track CCHCR1 redistribution using FITC-conjugated antibody

  • Compare with known P-body component redistribution patterns

• Peptide competition assay:

  • Pre-incubate FITC-CCHCR1 antibody with immunizing peptide

  • Apply to cells in parallel with non-blocked antibody

  • Quantify signal reduction specifically in P-bodies

This comprehensive validation approach ensures that FITC-CCHCR1 antibody signals in P-bodies represent specific detection rather than artifacts, enabling reliable mechanistic studies of CCHCR1's role in RNA metabolism.

What are the technical challenges in using FITC-conjugated CCHCR1 antibodies for flow cytometry?

Flow cytometric analysis using FITC-conjugated CCHCR1 antibodies presents several technical challenges that require methodological solutions to ensure data reliability and interpretability. These issues arise from both antibody characteristics and biological properties of CCHCR1.

Technical Challenges and Solutions:

• Signal intensity optimization:

  • Challenge: CCHCR1 expression levels vary significantly across cell types and cell cycle stages

  • Solution:

    • Implement titration experiments (typically 1:10-1:50 dilutions)

    • Standard protocol: 2 μL antibody per 1×10^6 cells in 100 μL final volume

    • Compare signal with positive control cells (e.g., HEK-293 with confirmed expression)

• Autofluorescence management:

  • Challenge: FITC emission overlaps with cellular autofluorescence

  • Solution:

    • Include unstained controls for each cell type

    • Implement compensation controls if multiplexing

    • Consider alternative detection antibodies in AF488 channel if autofluorescence is prohibitive

• Subcellular localization constraints:

  • Challenge: CCHCR1 localizes to nuclear/P-body/centrosome compartments, potentially limiting antibody access

  • Solution:

    • Optimize permeabilization protocols (test Triton X-100, saponin, methanol)

    • Compare different fixation methods (formaldehyde vs. methanol)

    • Ensure sufficient incubation time for antibody penetration

• Specificity verification:

  • Challenge: Potential cross-reactivity with similar proteins

  • Solution:

    • Include isotype control antibodies conjugated to FITC

    • Compare staining in CCHCR1-positive vs. CCHCR1-negative cells

    • Use peptide competition controls

    • Test non-specificity on non-target cells (e.g., CD3+ cells in human PBMCs)

• Cell cycle-dependent expression:

  • Challenge: CCHCR1 is regulated by E2F1 with expression peaking at G1/S transition

  • Solution:

    • Consider cell cycle synchronization before analysis

    • Co-stain with cell cycle markers (e.g., PCNA, Ki-67)

    • Analyze CCHCR1 expression in cell cycle-gated populations

• Protocol standardization:

  Parameter	Recommended Approach
  Cell number	1-5×10^6 cells per sample
  Antibody amount	2 μL stock per 10^6 cells (adjust based on titration)
  Incubation	30 minutes at 2-8°C, protected from light
  Washing	3× with cold PBS + 2% FBS
  Final volume	200-400 μL in PBS + 2% FBS

By addressing these technical challenges systematically, researchers can obtain reliable flow cytometric data on CCHCR1 expression and distribution across different cell populations and experimental conditions.

How might the bidirectional promoter between CCHCR1 and TCF19 impact experimental design when studying cell cycle regulation?

The bidirectional promoter architecture shared between CCHCR1 and TCF19 presents unique experimental design considerations for cell cycle regulation studies. This genomic arrangement, where both genes are co-regulated by E2F1 during G1/S transition , necessitates sophisticated methodological approaches.

Experimental Design Considerations:

• Promoter analysis strategies:

  • The 287 bp intergenic sequence serves as minimal promoter for both genes

  • CCHCR1 expression enhancement requires exon 1 sequences from both genes

  • Recommended reporter constructs:

    • Bidirectional luciferase reporters with intergenic region alone

    • Constructs incorporating exon 1 from each gene

    • E2F1 binding site mutants to confirm regulation mechanism

• Gene manipulation approaches:

  Strategy	Advantages	Limitations	Implementation
  siRNA knockdown	Simple delivery	Potential off-target effects	Target unique 3' UTR regions
  CRISPR/Cas9 editing	Precise targeting	Challenging delivery	Edit coding sequences rather than promoter
  Inducible expression	Temporal control	System leakiness	Use heterologous promoters

• Cell synchronization methods for expression analysis:

  • Double thymidine block for S-phase enrichment

  • Nocodazole block/release for M/G1 transition

  • Serum starvation/stimulation for G0/G1/S progression

  • Protocol optimization:

    • Validate synchronization by flow cytometry

    • Collect samples at multiple timepoints (e.g., 0, 2, 4, 8, 12, 24h)

    • Monitor both CCHCR1 and TCF19 expression simultaneously

• E2F1 regulatory context evaluation:

  • Combine E2F1 ChIP with expression analysis

  • Test effects of E2F1 overexpression/knockdown on both genes

  • Assess compensation by other E2F family members

  • Correlate with Rb pathway status and cyclin/CDK activity

• Data interpretation framework:

  • Consider multiple causal relationships:

    • Direct effects of target gene manipulation

    • Indirect effects via altered partner gene expression

    • Combined effects from both genes

  • Essential controls:

    • Single gene rescue in dual knockdown background

    • Heterologous expression systems lacking bidirectional control

    • Correlation with endogenous cell cycle markers

This specialized experimental design framework accounts for the unique genomic arrangement of CCHCR1 and TCF19, enabling more accurate characterization of CCHCR1's cell cycle-dependent regulation and function.

What are the potential pitfalls in interpreting FITC-conjugated CCHCR1 antibody signals in psoriasis tissue samples?

Interpretation Challenges and Methodological Solutions:

• Tissue autofluorescence interference:

  • Challenge: Psoriatic lesions exhibit elevated autofluorescence due to hyperkeratosis and inflammatory infiltrates

  • Solutions:

    • Include unstained adjacent sections as autofluorescence controls

    • Apply spectral unmixing algorithms if available

    • Consider alternative fluorophores (longer wavelengths) if autofluorescence overwhelms FITC signal

    • Implement tissue autofluorescence quenching procedures

• Antigen retrieval complexities:

  • Challenge: Psoriatic tissue architecture affects epitope accessibility

  • Solutions:

    • Compare TE buffer pH 9.0 and citrate buffer pH 6.0 retrieval methods

    • Optimize retrieval duration for psoriatic specimens

    • Consider protease-assisted retrieval for heavily cross-linked samples

    • Validate epitope recovery with positive control tissues

• Expression heterogeneity across lesions:

  • Challenge: CCHCR1 expression varies between lesional regions and disease stages

  • Solutions:

    • Implement systematic sampling across multiple lesional regions

    • Develop quantitative scoring systems (e.g., H-score or automated intensity measurement)

    • Include clinical stratification (acute vs. chronic, treated vs. untreated)

    • Apply tissue microarray approaches for standardized comparison

• Cellular differentiation effects:

  Epidermal Layer	Expected CCHCR1 Pattern	Methodological Consideration
  Basal layer	Potential proliferation-associated expression	Compare with Ki-67 co-staining
  Spinous layer	Variable expression	Correlate with differentiation markers
  Granular layer	Potentially altered in psoriasis	Compare with normal skin patterns
  Cornified layer	Autofluorescence interference	Implement specific background correction

• Immune infiltrate considerations:

  • Challenge: Inflammatory cells may express CCHCR1 or cause non-specific binding

  • Solutions:

    • Co-stain with immune cell markers (CD3, CD4, CD8, CD11c)

    • Use confocal microscopy to distinguish epidermal vs. immune cell staining

    • Include isotype controls at equivalent concentrations

    • Validate with double immunofluorescence

• Comparative analysis framework:

  • Challenge: Comparing lesional vs. non-lesional skin requires standardization

  • Solutions:

    • Process paired samples simultaneously

    • Use identical acquisition settings

    • Implement internal reference standards

    • Apply quantitative image analysis with defined regions of interest

By systematically addressing these pitfalls, researchers can obtain more reliable interpretations of FITC-conjugated CCHCR1 antibody signals in psoriatic tissues, facilitating accurate assessment of CCHCR1's role in this inflammatory skin condition.

How can ChIP assays be optimized when using FITC-conjugated CCHCR1 antibodies?

Chromatin immunoprecipitation (ChIP) using FITC-conjugated CCHCR1 antibodies presents unique challenges requiring specialized optimization strategies. While FITC conjugation is primarily designed for detection applications rather than ChIP, researchers may encounter scenarios requiring this approach.

Optimization Strategy for CCHCR1 ChIP:

• Antibody selection and validation:

  • Challenge: FITC conjugation may interfere with epitope recognition during ChIP

  • Solutions:

    • Compare ChIP efficiency between conjugated and unconjugated antibodies

    • Validate antibodies targeting different epitopes (AA 281-500, AA 483-782, AA 599-627)

    • Confirm antibody specificity via western blot prior to ChIP

    • Consider antibody concentration adjustments to compensate for FITC effects

• Cross-linking optimization:

  • Challenge: CCHCR1 may interact with chromatin indirectly through protein partners

  • Solutions:

    • Implement dual cross-linking approach:

      • Primary formaldehyde (1%) fixation: 10 minutes at room temperature

      • Secondary protein cross-linker (DSG, EGS): 20 minutes prior to formaldehyde

    • Optimize cross-linking time through time-course experiments

    • Consider native ChIP for stable interactions

• Chromatin preparation protocol:

  Parameter	Standard Protocol	CCHCR1-Specific Optimization
  Cell number	2×10^6 cells per IP	Increase to 5-10×10^6 cells for lower abundance protein
  Sonication	8-12 cycles (30s on/30s off)	Optimize for cell type; confirm by agarose gel
  Fragment size	200-500 bp	Target 200-300 bp for higher resolution
  Pre-clearing	1-2 hours with beads	Extended pre-clearing (3-4 hours) to reduce background

• Immunoprecipitation conditions:

  • Challenge: Potential lower affinity of FITC-conjugated antibodies

  • Solutions:

    • Increase antibody amount (3-5 μg per IP)

    • Extend incubation time (overnight at 4°C with rotation)

    • Test anti-FITC secondary antibodies for immune complex formation

    • Optimize buffer conditions (test RIPA vs. NP-40-based buffers)

• Beads selection and processing:

  • Protein A/G mixture for rabbit polyclonal antibodies

  • Magnetic beads for improved recovery and reduced background

  • Multiple stringent washes to reduce non-specific binding:

    • Low salt buffer (150 mM NaCl)

    • High salt buffer (500 mM NaCl)

    • LiCl buffer (250 mM LiCl)

    • TE buffer

• Target region selection based on CCHCR1 biology:

  • Bidirectional promoter shared with TCF19 (287 bp intergenic region)

  • E2F1-regulated promoter elements

  • Exon 1 regions that enhance expression

• Data analysis and validation:

  • qPCR with carefully designed primers for target regions

  • Multiple reference genes for normalization

  • ChIP-seq consideration for genome-wide binding analysis

  • Biological replicates (minimum n=3) for statistical validation

This comprehensive optimization strategy addresses the unique challenges of performing ChIP with FITC-conjugated CCHCR1 antibodies, enabling investigation of CCHCR1's potential chromatin associations during cell cycle regulation.