CCHCR1 Antibody, HRP conjugated

What are the primary applications for CCHCR1 antibody, HRP conjugated?

CCHCR1 antibody with HRP conjugation is optimized for several detection methods including Western Blotting (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry on paraffin-embedded tissues (IHC-P), and Immunohistochemistry on frozen sections (IHC-F) . The HRP (Horseradish Peroxidase) conjugation eliminates the need for secondary antibodies, streamlining experimental workflows while maintaining sensitivity. For Western Blotting applications, dilution ranges of 1:300-5000 are typically recommended, while ELISA applications generally require 1:500-1000 dilutions . For IHC-P and IHC-F, optimal dilution ranges are 1:200-400 and 1:100-500, respectively . Researchers should validate these dilutions for their specific experimental system.

How should CCHCR1 antibody, HRP conjugated be stored to maintain activity?

For optimal performance, CCHCR1 antibody with HRP conjugation should be stored at -20°C . The antibody is typically supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol . To prevent activity loss from repeated freeze-thaw cycles, it is strongly recommended to aliquot the antibody into multiple vials before freezing . This practice preserves the enzymatic activity of the HRP moiety, which is critical for detection sensitivity in all applicable methods.

What is the reactivity spectrum of commercially available CCHCR1 antibodies?

Commercial CCHCR1 antibodies show confirmed reactivity with human and rat CCHCR1 proteins . Additionally, they are predicted to cross-react with mouse, dog, cow, horse, and rabbit CCHCR1, though this cross-reactivity should be experimentally validated before use in these species . When selecting a CCHCR1 antibody for your research, it's important to match the antibody's documented reactivity with your experimental model organism. Some antibodies may recognize specific amino acid regions (such as AA 483-782 or AA 281-500) rather than the full-length protein, so consider whether your target protein variant contains these regions .

How can I validate the specificity of CCHCR1 antibody for all three protein isoforms?

Validating CCHCR1 antibody specificity for all three isoform variants requires a systematic approach. First, determine if your antibody's epitope is contained within regions common to all isoforms. The antibody described in the search results recognizes an epitope within amino acids 31-130/243, which is present in all three isoform variants . For proper validation, perform Western blotting with positive controls expressing each isoform separately, such as recombinant proteins or cell lines with confirmed expression. Employ both positive and negative controls, including CCHCR1 knockout/knockdown samples. To further confirm specificity, pre-absorption tests can be conducted by pre-incubating the antibody with its immunizing peptide before application, which should abolish specific signals. Mass spectrometry analysis of immunoprecipitated proteins can provide additional confirmation of antibody specificity across all isoforms.

What factors might affect CCHCR1 detection in subcellular localization studies?

Several factors can impact CCHCR1 detection in subcellular localization studies. CCHCR1 has been reported in multiple cellular compartments, including the cytoplasm, nucleus, mitochondria, centrosome, and notably, in P-bodies as part of mRNA metabolism complexes . The protein's localization may vary depending on cell type, activation state, and potentially disease conditions. When conducting immunofluorescence or subcellular fractionation experiments, consider that epitope accessibility might differ between applications and fixation methods. The N-terminus of CCHCR1 is specifically required for its localization to P-bodies , so antibodies targeting different regions may yield varying results. Cell permeabilization conditions should be optimized, as overly harsh treatments may disrupt P-body structures. Additionally, use colocalization with known P-body markers such as EDC4 to confirm CCHCR1 localization . Always include appropriate positive controls, such as cell lines with confirmed CCHCR1 expression patterns.

How can I optimize double staining protocols using CCHCR1 antibody, HRP conjugated with other markers?

Optimizing double staining protocols with HRP-conjugated CCHCR1 antibody and other markers requires careful planning to avoid signal interference. For immunohistochemistry applications with multiple markers, sequential detection is recommended over simultaneous application. Begin by staining for the less abundant antigen using the HRP-conjugated CCHCR1 antibody. After developing with an appropriate substrate (DAB yields a brown precipitate), perform thorough washing steps followed by blocking with hydrogen peroxide to quench remaining HRP activity. The second marker should be detected using an alternative enzyme system such as alkaline phosphatase with a contrasting substrate color. For fluorescence-based multiplexing, consider using a primary unconjugated CCHCR1 antibody instead, followed by fluorophore-conjugated secondary antibodies with spectrally distinct emission profiles. If using the HRP-conjugated antibody is necessary, tyramide signal amplification (TSA) methods can allow for multiplexing by removing the primary-secondary antibody complexes while preserving the deposited fluorophores between rounds of staining.

How does CCHCR1 expression in skin cancers differ from its expression in psoriasis?

CCHCR1 exhibits distinct expression patterns in skin cancers compared to psoriasis, which may inform experimental design and interpretation. In squamous cell carcinoma (SCC), CCHCR1 protein is predominantly localized at the pushing border of tumors, while in basal cell carcinoma (BCC), it lines the tumor islands . A key distinction from psoriasis is the correlation with proliferation markers: unlike in psoriasis, CCHCR1 expression in skin tumors shows a similar pattern to Ki67 proliferation marker . The most intense CCHCR1 staining in tumors occurs in areas also positive for epidermal growth factor receptor (EGFR) . At the molecular level, CCHCR1 mRNA is upregulated by 30-80% in SCC cell lines compared to normal keratinocytes, positively correlating with Ki67 expression . Interestingly, more aggressive and invasive tumor cell lines (RT3, FaDu) express CCHCR1 mRNA at lower levels than non-tumorigenic HaCaT cells . This suggests a complex relationship between CCHCR1 expression and cancer progression, where it may function as a negative regulator of proliferation in early stages of keratinocyte transformation but loses this control function in advanced oncogenesis .

What is the significance of CCHCR1's interaction with EDC4 in P-bodies for experimental design?

The interaction between CCHCR1 and EDC4 in P-bodies has significant implications for experimental design when studying mRNA metabolism and post-transcriptional regulation. P-bodies are cytoplasmic foci involved in mRNA decay and storage, and EDC4 (enhancer of mRNA-decapping protein 4) is a key component of these structures . When designing experiments to study CCHCR1, researchers should consider co-immunoprecipitation assays to verify this interaction in their specific cell types of interest. Confocal microscopy with co-staining for CCHCR1 and P-body markers (including EDC4) is essential to confirm localization . The N-terminus of CCHCR1 is required for its P-body localization, so deletion constructs or antibodies targeting different domains may yield different localization patterns . RNA immunoprecipitation (RIP) or crosslinking immunoprecipitation (CLIP) assays can help identify mRNAs associated with CCHCR1-containing complexes. For functional studies, consider designing experiments that assess changes in mRNA stability, decay rates, or translational efficiency following CCHCR1 modulation. Since disruption of CCHCR1 function may affect the cellular transcriptome, RNA-Seq experiments comparing wild-type to CCHCR1-depleted cells can provide valuable insights into its physiological roles .

How can CCHCR1 antibodies be utilized to investigate its role in proliferation regulation?

To investigate CCHCR1's role in proliferation regulation, researchers can employ multiple antibody-based approaches. Immunohistochemistry with CCHCR1 antibodies can be performed alongside proliferation markers (Ki67, PCNA) to evaluate their correlation in various tissue contexts. Since CCHCR1 is suggested to function as a negative regulator of proliferation in both psoriasis and early stages of keratinocyte transformation , experiments should be designed to manipulate CCHCR1 levels (overexpression or knockdown) followed by proliferation assays. For cell cycle analysis, combine CCHCR1 immunostaining with flow cytometry to determine if CCHCR1 levels fluctuate during different cell cycle phases. Co-immunoprecipitation with CCHCR1 antibodies can identify proliferation-related binding partners beyond the known EDC4 interaction . Chromatin immunoprecipitation (ChIP) may be useful if CCHCR1 influences gene expression through chromatin association. Additionally, since tumor promoters like okadaic acid and menadione downregulate CCHCR1 mRNA , researchers can investigate the molecular mechanisms behind this regulation using reporter assays with CCHCR1 promoter constructs. Cell fractionation followed by Western blotting with CCHCR1 antibodies can track its subcellular redistribution in response to proliferative signals or stress conditions.

What are common issues when using CCHCR1 antibody, HRP conjugated in Western blotting applications?

When using HRP-conjugated CCHCR1 antibody in Western blotting, several technical challenges may arise. High background signal is often encountered, which can be mitigated by optimizing antibody dilution (starting with the recommended 1:300-5000 range ), increasing blocking stringency, or extending washing durations. If detecting endogenous CCHCR1, sensitivity issues may occur due to low expression levels; in such cases, increasing protein loading (up to 50-80 μg per lane) or employing enhanced chemiluminescence (ECL) substrates can improve detection. Multiple bands may appear due to CCHCR1's alternative splice variants or post-translational modifications; validate these by comparison with positive control lysates expressing specific isoforms. The direct HRP conjugation eliminates secondary antibody cross-reactivity issues but may result in reduced signal amplification compared to two-step detection systems. If signal is weak, consider longer exposure times or using a more sensitive substrate. For quantitative Western blotting, strip and reprobe membranes with loading control antibodies, ensuring the HRP activity of the CCHCR1 antibody is fully quenched with hydrogen peroxide before applying the second primary antibody.

How can I minimize non-specific staining when using CCHCR1 antibody, HRP conjugated in immunohistochemistry?

Minimizing non-specific staining with HRP-conjugated CCHCR1 antibody in immunohistochemistry requires a systematic optimization approach. Begin with proper tissue fixation and processing, as overfixation can mask epitopes while underfixation may compromise tissue morphology. Implement heat-induced epitope retrieval (HIER) with citrate (pH 6.0) or EDTA (pH 9.0) buffers, testing both to determine optimal conditions for CCHCR1 detection. Thorough blocking is crucial; use 3-5% normal serum from the same species as the secondary antibody would be (if using an unconjugated primary), or 1-3% BSA with 0.1-0.3% Triton X-100 for the HRP-conjugated antibody. Optimize the antibody concentration using a dilution series around the recommended ranges (1:200-400 for IHC-P and 1:100-500 for IHC-F ). Extend the wash steps between incubations, using TBS-T with gentle agitation. Include proper controls: (1) no primary antibody, (2) isotype control, and (3) positive control tissues with known CCHCR1 expression. To reduce endogenous peroxidase activity, pretreat tissues with 0.3-3% hydrogen peroxide in methanol before antibody application. If background persists, consider pre-adsorption of the antibody with the immunizing peptide to confirm specificity.

What optimization strategies are recommended for CCHCR1 detection in skin cancer tissue samples?

For optimal CCHCR1 detection in skin cancer tissue samples, several specialized strategies should be considered based on the unique characteristics of skin tumors and CCHCR1 expression patterns. Since CCHCR1 is most intensely expressed at tumor pushing borders and in areas positive for EGFR , careful attention to these regions during tissue selection and processing is essential. For formalin-fixed paraffin-embedded (FFPE) samples, limit fixation time to 12-24 hours to prevent overfixation, which can mask epitopes. During antigen retrieval, test both citrate and EDTA-based buffers, as CCHCR1 epitopes may respond differently in the context of tumor tissue. Dual staining with proliferation markers like Ki67 can provide valuable context, as CCHCR1 and Ki67 show similar expression patterns in skin tumors (unlike in psoriasis) . To differentiate between tumor and stromal staining, consider multiplex immunohistochemistry with epithelial markers. Given the observed correlation between CCHCR1 and EGFR expression in tumors , double staining for both proteins may yield insights into their functional relationship. Control for melanin, which can mimic DAB-based HRP detection, by using alternative chromogens or performing melanin bleaching before immunostaining. For quantitative analysis, use digital pathology approaches with appropriate software to measure staining intensity specifically in tumor regions versus surrounding stroma.

How should researchers interpret differences in CCHCR1 staining patterns between normal tissue and carcinomas?

When interpreting differences in CCHCR1 staining patterns between normal tissue and carcinomas, researchers should consider several key aspects of CCHCR1 biology and tumor pathology. In normal skin, CCHCR1 expression is typically limited and regulated, while in carcinomas, CCHCR1 shows upregulation with specific localization patterns—particularly at pushing borders of squamous cell carcinoma and lining basal cell carcinoma islands . Quantitative assessment should include both staining intensity and percentage of positive cells, preferably using digital image analysis for objectivity. The co-localization of CCHCR1 with EGFR is particularly significant and may indicate coordinated pathway activation . Unlike in psoriasis, CCHCR1 in tumors shows correlation with proliferation marker Ki67, suggesting context-dependent functions . Researchers should note that while CCHCR1 mRNA is upregulated 30-80% in SCC lines compared to normal keratinocytes, more aggressive tumor cell lines express lower levels than non-tumorigenic cells , indicating a potential biphasic relationship with tumor progression. This paradoxical finding suggests CCHCR1 may function as a negative regulator of proliferation in early oncogenesis but loses this control in advanced stages . Analysis should also account for tumor heterogeneity, as CCHCR1 expression may vary across different regions of the same tumor.

What controls should be included when validating CCHCR1 antibody specificity in cancer research applications?

A comprehensive validation strategy for CCHCR1 antibody specificity in cancer research should include multiple controls addressing both technical and biological variables. Technical controls should include: (1) No primary antibody control to assess secondary antibody non-specific binding or endogenous peroxidase activity; (2) Isotype control using non-specific IgG at the same concentration as the CCHCR1 antibody; (3) Peptide competition/neutralization assay using the immunizing peptide to confirm binding specificity. Biological controls should include: (1) CCHCR1 knockdown/knockout cell lines or tissues to confirm signal reduction; (2) Positive control tissues with known CCHCR1 expression, such as psoriatic skin; (3) Western blot correlation with immunohistochemistry results to confirm detection of the expected protein size; (4) Comparison of staining with multiple CCHCR1 antibodies targeting different epitopes. For cancer-specific applications, include normal adjacent tissue within tumor samples as internal controls. Cross-validate with mRNA expression data through methods like in situ hybridization or RT-qPCR from parallel samples. Additionally, since CCHCR1 is associated with P-bodies , co-staining with P-body markers in cultured cancer cells can provide functional validation of the antibody's ability to recognize CCHCR1 in its native complex.

How does the subcellular localization of CCHCR1 correlate with its proposed functions in different cellular contexts?

The subcellular localization of CCHCR1 provides critical insights into its diverse functions across different cellular contexts. CCHCR1 has been reported in multiple compartments, including the cytoplasm, nucleus, mitochondria, centrosome, and P-bodies , with each localization potentially reflecting distinct functional roles. The identification of CCHCR1 in P-bodies through its interaction with EDC4 suggests involvement in mRNA metabolism and post-transcriptional regulation . This localization depends specifically on the N-terminus of CCHCR1 , indicating domain-specific functional roles. In skin cancer contexts, CCHCR1's correlation with EGFR expression may indicate participation in growth factor signaling pathways, possibly through regulation of mRNA stability for pathway components. The similar distribution patterns of CCHCR1 and Ki67 in skin tumors (but not in psoriasis) suggests context-dependent functions in proliferation regulation. When analyzing CCHCR1 subcellular localization, researchers should employ co-localization studies with markers for each potential compartment using high-resolution confocal microscopy, and validate findings with biochemical fractionation followed by Western blotting. Changes in CCHCR1 localization during cell cycle progression, differentiation, or in response to stress stimuli may reveal dynamic regulation mechanisms. Additionally, localization patterns may differ between normal and pathological states, providing potential diagnostic or prognostic biomarkers.

How can phosphoproteomic analysis be combined with CCHCR1 immunoprecipitation to investigate its signaling functions?

Combining phosphoproteomic analysis with CCHCR1 immunoprecipitation provides a powerful approach to decipher its signaling functions and regulatory network. To implement this strategy, begin with immunoprecipitation using anti-CCHCR1 antibodies from cells in different states (normal vs. stimulated with growth factors, stress conditions, or disease models). For comprehensive analysis, perform parallel immunoprecipitations: one for CCHCR1 itself to identify interacting partners, and another for phosphoproteins associated with CCHCR1 complexes. The precipitated proteins should undergo tryptic digestion followed by phosphopeptide enrichment using titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC). Analyze enriched phosphopeptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electron transfer dissociation (ETD) or higher-energy collisional dissociation (HCD) for optimal phosphosite identification . Computational analysis of the resulting phosphoproteome can reveal kinase motifs overrepresented in CCHCR1-associated proteins, indicating potential upstream regulators. Validate key findings using phospho-specific antibodies against identified sites in Western blot analysis. To establish causality, combine this approach with kinase inhibitor treatment or phosphatase overexpression to determine how modulating specific phosphorylation events affects CCHCR1's interaction network, subcellular localization (particularly in P-bodies), and functional outcomes like mRNA stability or cellular proliferation.

What specialized techniques are required to study CCHCR1's role in P-body dynamics and mRNA metabolism?

Investigating CCHCR1's role in P-body dynamics and mRNA metabolism requires specialized techniques spanning molecular, cellular, and biochemical approaches. Begin with live-cell imaging using fluorescently-tagged CCHCR1 and established P-body markers (like EDC4 ) to track P-body assembly, disassembly, and mobility in real-time under various cellular conditions. FRAP (Fluorescence Recovery After Photobleaching) can assess CCHCR1 mobility and exchange rates within P-bodies. To identify mRNAs regulated by CCHCR1, perform RNA-immunoprecipitation (RIP) or crosslinking immunoprecipitation (CLIP) followed by high-throughput sequencing to map CCHCR1-RNA interactions transcriptome-wide. Proximity labeling techniques such as BioID or APEX can identify proteins in close spatial proximity to CCHCR1 within P-bodies, potentially revealing functional modules. For functional studies, establish CCHCR1 knockout/knockdown systems and measure effects on global mRNA decay rates using actinomycin D chase experiments combined with RNA-Seq. Since the N-terminus of CCHCR1 is required for P-body localization , use domain mapping with truncation mutants to identify specific motifs mediating interactions with P-body components. To understand regulation, analyze how stress conditions known to affect P-body dynamics (oxidative stress, heat shock, arsenite treatment) impact CCHCR1 localization and function. Polysome profiling can assess how CCHCR1 manipulation affects mRNA translation states, connecting its role in mRNA metabolism to translational outcomes.

How might single-cell analysis methods enhance our understanding of CCHCR1 heterogeneity in tumor microenvironments?

Single-cell analysis methods offer unprecedented insights into CCHCR1 heterogeneity within tumor microenvironments, revealing cell-specific expression patterns and functional states not detectable with bulk tissue analyses. Single-cell RNA sequencing (scRNA-seq) of tumor samples can profile CCHCR1 expression across diverse cell populations, identifying specific cancer cell subpopulations or stromal cells with distinctive CCHCR1 expression levels. This approach can be coupled with trajectory analysis to map how CCHCR1 expression changes during tumor evolution or in response to therapy. For protein-level analysis, mass cytometry (CyTOF) with metal-conjugated CCHCR1 antibodies enables simultaneous detection of dozens of other proteins, allowing correlation of CCHCR1 with cell state markers, signaling phosphoproteins, and other tumor microenvironment features. Multiplex immunofluorescence imaging preserves spatial context, critical for understanding CCHCR1's distribution at tumor pushing borders and its relationship with neighboring cells. Single-cell Western blotting can validate protein isoform expression in rare cell populations. For functional analysis, CRISPR-based lineage tracing combined with CCHCR1 reporter systems can track the fate of CCHCR1-expressing cells during tumor progression. Digital spatial profiling allows region-specific quantification of CCHCR1 alongside hundreds of other proteins or RNAs, particularly valuable given CCHCR1's association with EGFR-positive regions in tumors . These approaches collectively provide a multidimensional view of CCHCR1 biology in the complex ecosystem of tumors, potentially revealing new therapeutic vulnerabilities or biomarker applications.