HIC1 Antibody, HRP conjugated

What is HIC1 and why is it significant in cancer research?

HIC1 (Hypermethylated in Cancer 1) was originally identified as a target of p53-induced gene expression and functions as a putative tumor suppressor protein that mediates transcriptional repression. The significance of HIC1 in cancer research stems from its frequent suppression in leukemia and various cancers due to hypermethylation of specific DNA regions, resulting in transcriptional silencing . HIC1 is also deleted in the genetic disorder Miller-Dieker syndrome (MDS), further highlighting its biological importance . Structurally, HIC1 is defined by five zinc fingers and an N-terminal broad complex POZ (or BTB) domain, which interacts with the SMRT/N-CoR-mSin3A HDAC complex to repress gene transcription .

What are the key specifications of commercially available HIC1 Antibody, HRP conjugated?

The HIC1 polyclonal antibody with HRP conjugation (such as bs-15485R-HRP) is designed with the following specifications:

These specifications ensure optimal performance in multiple experimental applications while maintaining antibody stability .

How does the subcellular localization of HIC1 influence antibody selection and experimental design?

HIC1 primarily localizes to the nucleus where it functions as a transcriptional repressor . When visualized by immunofluorescence microscopy, endogenous HIC1 proteins appear in punctate nuclear structures, characteristic of proteins containing a BTB/POZ domain . This distinct nuclear localization pattern necessitates careful antibody selection and experimental design considerations:

• Nuclear extraction protocols should be optimized to efficiently isolate HIC1 for Western blot analysis

• For immunohistochemistry, appropriate antigen retrieval methods are critical - both TE buffer (pH 9.0) and citrate buffer (pH 6.0) have been successfully employed

• When selecting controls for specificity, siRNA knockdown approaches can confirm antibody specificity, as demonstrated in studies where HIC1-positive nuclear dots were not detectable in cells transfected with HIC1-specific siRNA

• For co-localization studies, nuclear markers should be incorporated to confirm proper detection of HIC1's punctate nuclear pattern

Understanding this subcellular localization is essential for accurate interpretation of experimental results with HIC1 antibodies .

What are the optimal dilution ranges for different applications of HIC1 Antibody?

Based on validated experimental protocols, the following dilution ranges are recommended for HIC1 antibodies in various applications:

It is strongly recommended that researchers titrate the antibody in their specific testing systems to determine optimal concentrations for their particular experimental conditions . The observed molecular weight of HIC1 is typically 65-70 kDa, which is slightly lower than the calculated molecular weight of 75 kDa (714 amino acids) . This discrepancy should be considered when interpreting Western blot results.

What controls should be included when validating HIC1 antibody specificity?

Rigorous validation of HIC1 antibody specificity requires several types of controls:

• Positive controls: Jurkat cells and NIH/3T3 cells have been validated for Western blot applications . For tissue sections, rat lung and rat stomach tissues have shown positive IHC detection .

• Negative controls:

  • Primary antibody omission: Samples incubated with secondary antibodies only should show no specific staining

  • siRNA knockdown: WPMY-1 cells transfected with HIC1-specific siRNA showed elimination of HIC1 immunoreactivity, confirming antibody specificity

• Expression validation controls:

  • RT-qPCR analysis to confirm HIC1 expression levels in test samples

  • Comparison with known HIC1 expression patterns (e.g., higher expression in stromal vs. epithelial compartments)

• Cross-reactivity assessment:

  • Testing in multiple cell lines with different HIC1 expression levels (e.g., WPMY-1 myofibroblasts vs. RWPE1 epithelial cells)

  • Inclusion of recombinant HIC1 protein as a positive control

Implementation of these controls ensures reliable and reproducible results when working with HIC1 antibodies .

How should HIC1 antibody be stored and handled to maintain optimal activity?

To maintain optimal HIC1 antibody activity, adhere to these storage and handling guidelines:

• Storage temperature: Store at -20°C for long-term preservation

• Aliquoting strategy:

  • Divide the stock solution into small aliquots to avoid repeated freeze-thaw cycles

  • This is particularly important for HRP-conjugated antibodies, which can lose activity with repeated freezing and thawing

• Buffer composition:

  • The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) or aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol

  • These components help maintain antibody stability

• Stability period:

  • Most antibodies remain stable for one year after shipment when stored properly

  • For 20μl sizes, the presence of 0.1% BSA aids in stability

• Working solution handling:

  • Prepare fresh dilutions on the day of the experiment

  • Keep diluted antibody cold and protected from light, especially HRP-conjugated versions

  • Avoid contamination by using clean pipette tips and sterile containers

Following these guidelines will help ensure consistent and reliable results across experiments .

How can HIC1 antibody be used to investigate HIC1's role in transcriptional regulation networks?

HIC1 functions as a transcriptional repressor involved in regulatory loops modulating P53-dependent and E2F1-dependent cell survival, growth control, and stress responses . To investigate these complex networks:

• Chromatin Immunoprecipitation (ChIP) approaches:

  • HIC1 antibody can be used to identify direct binding sites on target genes

  • Studies have demonstrated HIC1 binding to target genes such as Cyclin D1 and p57KIP2 in quiescent WI38 cells, but not in growing cells

  • ChIP-seq analysis can provide genome-wide binding profiles to identify novel HIC1 targets

• Co-immunoprecipitation to identify protein interaction partners:

  • HIC1 interacts with corepressors including MTA1, a subunit of the NuRD complex

  • The BTB/POZ domain interacts with the SMRT/N-CoR-mSin3A HDAC complex in transcriptional repression

  • HRP-conjugated antibodies can be stripped and reprobedfor co-precipitated proteins

• Cellular context-dependent analysis:

  • Proliferation state affects HIC1 targeting (quiescent vs. growing cells show different binding patterns)

  • Cell-type specific regulation should be considered (e.g., differential expression in stromal vs. epithelial cells)

• Integration with epigenetic regulation studies:

  • Combine with DNA methylation analysis to correlate HIC1 binding with promoter methylation status

  • Assess histone modifications at HIC1 binding sites to understand chromatin context

This multifaceted approach can reveal how HIC1 coordinates transcriptional repression within broader regulatory networks .

How can expression patterns of HIC1 be accurately quantified across different tissue types?

Accurate quantification of HIC1 expression across tissue types requires complementary approaches:

• RT-qPCR optimization:

  • Design primers located in the large HIC1 coding exon 2 to amplify both major alternative HIC1 transcripts (HIC1 1a/variant 1 and HIC1 1b/variant 2)

  • Use appropriate reference genes validated for the specific tissue types being compared

  • Include appropriate controls (e.g., normal prostate from young healthy donors has served as a control in prostate cancer studies)

• Immunohistochemical quantification approaches:

  • Use recommended dilutions (1:500-1:2000) with optimized antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Employ digital image analysis with standardized scoring systems

  • Account for cell-type specific expression (e.g., in prostate, HIC1 shows stronger expression in stromal compared to epithelial compartments)

• Cell sorting for lineage-specific analysis:

  • FACS-sorting of specific cell populations prior to expression analysis can reveal lineage-specific patterns

  • Single-cell RNA-sequencing can identify HIC1 expression in distinct cell clusters within heterogeneous tissues

  • In prostate tissue, this approach revealed enrichment of HIC1 expressing cells in stromal compartments (fibroblasts, smooth muscle, endothelia, and leukocytes)

• Western blot quantification:

  • Use 65-70 kDa band for quantification (observed molecular weight)

  • Include loading controls appropriate for nuclear proteins

  • Compare expression across relevant cell lines as benchmarks (e.g., Jurkat, NIH/3T3)

This multi-method approach provides robust quantification of HIC1 expression patterns across diverse tissue types .

What strategies can resolve inconsistent HIC1 antibody results in cancer tissues?

Resolving inconsistent HIC1 antibody results in cancer tissues requires systematic troubleshooting:

• Tissue heterogeneity considerations:

  • Stromal content significantly affects HIC1 expression measurements in tumors

  • Some prostate tumors with high stromal content show elevated HIC1 expression compared to adjacent normal tissue, contrary to the typically observed downregulation

  • Microdissection or cell-type specific analysis may be necessary to account for this heterogeneity

• Technical optimization approaches:

  • Antigen retrieval method validation: Compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) to determine optimal conditions

  • Antibody concentration titration: Test multiple dilutions within the recommended range (1:500-1:2000)

  • Blocking optimization: Adjust blocking conditions to reduce background while preserving specific signal

• Multiple detection method validation:

  • Confirm IHC results with Western blot analysis

  • Validate protein-level findings with mRNA expression data

  • Use immunofluorescence to assess subcellular localization patterns

• Cancer stage and subtype stratification:

  • Stratify samples by TNM classification (e.g., pT3a and pT3b prostate tumors showed unique expression patterns)

  • Consider molecular subtypes that may influence HIC1 expression

  • Account for treatment history that may affect HIC1 expression or detection

This comprehensive approach can help reconcile apparently contradictory results and provide more accurate interpretation of HIC1 expression patterns in cancer tissues .

How does HIC1 expression change across cancer progression and what are the methodological considerations for accurate assessment?

HIC1 expression dynamics across cancer progression exhibit complex patterns requiring careful methodological considerations:

This multifaceted approach enables accurate tracking of HIC1 expression changes throughout cancer progression while accounting for tumor heterogeneity .

What are the technical considerations when using HIC1 antibody to study its interaction with regulatory proteins?

Studying HIC1 interactions with regulatory proteins requires specific technical considerations:

• Co-immunoprecipitation optimization:

  • Nuclear extraction protocols must be optimized to maintain protein-protein interactions

  • Crosslinking may be required to stabilize transient interactions

  • Salt concentration in washing buffers must be carefully titrated to preserve specific interactions while reducing background

• Proximity ligation assays (PLA):

  • Can be used to visualize and quantify HIC1 interactions with partners like MTA1

  • Requires careful antibody selection to ensure compatibility and specificity

  • Primary antibodies must be raised in different species for standard PLA protocols

• Cell state considerations:

  • HIC1 interactions vary with cellular context - quiescent versus proliferating cells show different interaction patterns

  • HIC1/MTA1 complexes bind target genes like Cyclin D1 and p57KIP2 in quiescent WI38 cells but not in growing cells

  • Synchronize cells appropriately to study context-dependent interactions

• Domain-specific interaction analysis:

  • HIC1's BTB/POZ domain interacts with the SMRT/N-CoR-mSin3A HDAC complex

  • Domain mapping experiments may require specific antibodies recognizing different HIC1 epitopes

  • Consider using epitope-tagged HIC1 constructs to overcome antibody limitations

• Interaction verification strategies:

  • Reciprocal co-immunoprecipitation with antibodies against interaction partners

  • siRNA-mediated knockdown of HIC1 or partner proteins to confirm specificity

  • Mass spectrometry to identify novel interaction partners in an unbiased manner

These technical considerations enable robust analysis of HIC1's interactions with its regulatory partners in different cellular contexts .

How can HIC1 antibodies be used to investigate the role of HIC1 in stromal-epithelial interactions in cancer?

HIC1 shows distinct expression patterns in stromal versus epithelial compartments, making it valuable for studying stromal-epithelial interactions in cancer:

• Dual immunofluorescence approaches:

  • Combine HIC1 antibody with epithelial markers (e.g., E-cadherin, cytokeratins) and stromal markers (e.g., α-SMA, vimentin)

  • This approach revealed HIC1 enrichment in stromal compartments of prostate tissue (fibroblasts, smooth muscle, endothelia, and leukocytes)

  • Quantitative analysis of co-localization can reveal spatial relationships between cell types

• Cell culture model systems:

  • Compare HIC1 expression in paired cell lines representing epithelial and stromal components of the same tissue (e.g., RWPE1 epithelial cells versus WPMY-1 myofibroblasts from prostate)

  • Co-culture systems can assess how stromal HIC1 expression influences epithelial cell behavior

  • Conditioned media experiments can identify secreted factors regulated by HIC1

• Laser capture microdissection with immunostaining:

  • Precision isolation of stromal and epithelial compartments following HIC1 immunostaining

  • RNA-seq or proteomics analysis of isolated compartments can reveal distinct gene expression profiles

  • Correlation of HIC1 expression with stromal markers in microdissected samples can confirm cell-type specific patterns

• Single-cell analysis integration:

  • Correlate HIC1 immunostaining with single-cell RNA-seq data from the same tissue type

  • This approach confirmed HIC1 enrichment in stromal cell clusters in prostate tissue

  • Spatial transcriptomics can provide additional context for HIC1 expression patterns

These approaches leverage HIC1 antibodies to investigate the complex interplay between stromal and epithelial compartments in cancer development and progression .

How can HIC1 antibodies be utilized in multiplex imaging systems for tumor microenvironment analysis?

Multiplex imaging with HIC1 antibodies offers powerful insights into tumor microenvironment dynamics:

• Cyclic immunofluorescence (CyCIF) integration:

  • HIC1 antibodies can be incorporated into CyCIF panels to visualize its expression alongside multiple cell type markers and signaling molecules

  • This approach can reveal spatial relationships between HIC1-expressing stromal cells and other components of the tumor microenvironment

  • HRP-conjugated antibodies would require tyramide signal amplification protocols optimized for multiplexing

• Mass cytometry imaging approaches:

  • Metal-tagged HIC1 antibodies enable highly multiplexed imaging via Imaging Mass Cytometry (IMC) or MIBI-TOF

  • These platforms allow simultaneous visualization of 40+ markers, enabling comprehensive characterization of HIC1-expressing cells within the complex tumor microenvironment

  • Antibody metal conjugation protocols must be optimized to preserve epitope recognition

• Spatial context quantification strategies:

  • Nearest neighbor analysis can quantify spatial relationships between HIC1+ cells and specific tumor or immune cell populations

  • Cell clustering algorithms can identify higher-order organizational patterns

  • These approaches can reveal whether HIC1+ stromal cells form specific niches within the tumor microenvironment

• Combined functional state assessment:

  • Multiplex panels can combine HIC1 with markers of cell proliferation, senescence, or activation

  • This can reveal functional heterogeneity within HIC1-expressing stromal populations

  • Integration with hypoxia markers can determine whether microenvironmental stress affects HIC1 expression patterns

These advanced multiplex imaging approaches provide unprecedented insights into the complex role of HIC1 in the tumor microenvironment .

What methodological considerations are important when studying HIC1 target genes using ChIP-seq with HIC1 antibodies?

ChIP-seq with HIC1 antibodies requires specific methodological considerations to generate reliable results:

• Antibody selection and validation:

  • Antibodies must be validated for ChIP applications specifically

  • Polyclonal antibodies may provide better coverage of epitopes that remain accessible in cross-linked chromatin

  • Pre-clearing steps may be necessary to reduce background with certain antibody preparations

• Chromatin preparation optimization:

  • Crosslinking conditions must be carefully optimized for nuclear transcription factors like HIC1

  • Sonication parameters should be adjusted to generate optimal fragment sizes (200-300bp)

  • Nuclear extraction protocols should be optimized to efficiently release chromatin-bound HIC1

• Context-dependent binding considerations:

  • Cell state significantly affects HIC1 binding patterns - quiescent versus proliferating cells show different target gene binding profiles

  • HIC1/MTA1 complexes bind targets like Cyclin D1 and p57KIP2 in quiescent but not growing cells

  • Synchronize cells appropriately to study context-dependent binding events

• Sequencing and analysis recommendations:

  • Include appropriate input controls for normalization

  • Consider the punctate nuclear distribution pattern of HIC1 when analyzing peak shapes

  • Motif analysis should account for both direct HIC1 binding sites and potential co-factor binding regions

  • Integration with transcriptome data can confirm functional significance of binding events

• Co-factor binding strategies:

  • Sequential ChIP (ChIP-reChIP) can identify genomic regions bound by both HIC1 and interacting partners like MTA1

  • This approach can distinguish different HIC1 complex compositions at different target genes

These methodological considerations enable robust ChIP-seq analysis of HIC1 binding sites and target genes under different cellular conditions .

How can phosphorylation status of HIC1 be assessed using modified immunodetection protocols?

Assessing HIC1 phosphorylation status requires specialized immunodetection protocols:

• Phosphatase inhibitor optimization:

  • Standard lysis buffers must be supplemented with phosphatase inhibitor cocktails optimized for nuclear proteins

  • Include sodium fluoride, sodium orthovanadate, sodium pyrophosphate, and β-glycerophosphate

  • Sample processing should be performed at 4°C to minimize dephosphorylation

• Phospho-specific antibody approaches:

  • While generic HIC1 antibodies detect total protein, phospho-specific antibodies would be required for direct phosphorylation assessment

  • In the absence of commercial phospho-specific antibodies, alternatives include:

    • Phospho-tag gel electrophoresis to separate phosphorylated from non-phosphorylated forms

    • Lambda phosphatase treatment of parallel samples to confirm phosphorylation-dependent mobility shifts

• Immunoprecipitation-based strategies:

  • Immunoprecipitate HIC1 using validated antibodies, then probe with anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibodies

  • Mass spectrometry analysis of immunoprecipitated HIC1 can identify specific phosphorylation sites

  • Compare phosphorylation patterns across different cellular conditions (quiescent vs. proliferating, normal vs. stressed)

• Functional correlation approaches:

  • Correlate detected phosphorylation states with HIC1 transcriptional repression activity

  • Assess co-factor binding (e.g., MTA1) in relation to phosphorylation status

  • Determine whether phosphorylation affects HIC1's punctate nuclear localization pattern

These specialized approaches enable comprehensive assessment of HIC1 phosphorylation status and its functional implications.

What are the critical quality control steps to ensure reproducible results with HIC1 antibodies across different research groups?

To ensure reproducible results with HIC1 antibodies across research groups, implement these critical quality control steps:

• Antibody validation documentation:

  • Maintain detailed records of antibody lot numbers, manufacturers, and validation experiments

  • Include antibody RRID (Research Resource Identifier) in publications (e.g., AB_2879816 for certain HIC1 antibodies)

  • Share validation protocols through repositories like Antibody Registry or protocols.io

• Standardized experimental protocols:

  • Develop and share detailed protocols for common applications (WB, IHC, ChIP)

  • Include specific buffer compositions, incubation times, and temperatures

  • Document antigen retrieval methods for IHC (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Consistent positive and negative controls:

  • Establish common positive control cell lines across labs (e.g., Jurkat cells, NIH/3T3 cells)

  • Use consistent negative control approaches (siRNA knockdown, primary antibody omission)

  • Consider developing reference standard materials for quantitative applications

• Data sharing and analysis standardization:

  • Share raw unprocessed images alongside analyzed data

  • Document image acquisition settings (exposure times, gain settings)

  • Use consistent quantification approaches and statistical methods

  • Consider automated analysis workflows to reduce subjective interpretation

• Multicenter validation studies:

  • Periodically conduct round-robin testing of the same samples across multiple laboratories

  • Share antibody aliquots from the same lot for critical comparative studies

  • Document environmental variables that might affect results (temperature, humidity)

Implementing these quality control measures will significantly enhance reproducibility of HIC1 antibody-based research across different laboratories.

What integrated approaches combining HIC1 antibody detection with other molecular techniques provide the most comprehensive understanding of HIC1 biology?

Comprehensive understanding of HIC1 biology requires integrated approaches combining antibody detection with complementary molecular techniques:

• Multi-omics integration strategies:

  • Combine HIC1 ChIP-seq with RNA-seq to correlate binding with transcriptional outcomes

  • Integrate DNA methylation profiling to assess epigenetic regulation of HIC1 and its target genes

  • Include proteomics analysis of HIC1 interactome under different cellular conditions

  • These approaches revealed complex regulation of targets like Cyclin D1 and p57KIP2 in quiescent versus growing cells

• Single-cell multi-parameter analysis:

  • Correlate single-cell RNA-seq with protein-level detection using imaging mass cytometry or similar platforms

  • This approach confirmed HIC1 enrichment in stromal cell clusters (fibroblasts, smooth muscle, endothelia, leukocytes) in prostate tissue

  • Extend to spatial transcriptomics to preserve tissue architecture context

• Functional genomics integration:

  • Combine CRISPR-Cas9 editing of HIC1 or its regulatory elements with antibody-based protein detection

  • Correlate phenotypic outcomes with molecular changes at protein and RNA levels

  • Include rescue experiments with wild-type and mutant HIC1 constructs to establish causality

• Structural biology connections:

  • Link antibody epitope mapping with structural studies of HIC1 domains

  • Particularly focus on the BTB/POZ domain that interacts with the SMRT/N-CoR-mSin3A HDAC complex

  • Use this information to develop more specific antibodies targeting functional domains

• Clinical-molecular correlations:

  • Integrate HIC1 antibody staining patterns in patient samples with clinical outcomes

  • Stratify by molecular subtypes, treatment history, and clinical parameters

  • This approach revealed unique expression patterns in advanced prostate tumors (pT3a and pT3b)

These integrated approaches provide a systems-level understanding of HIC1 biology across different cellular contexts and disease states .

How should researchers approach contradictory findings between HIC1 expression and functional outcomes in different experimental systems?

Resolving contradictory findings regarding HIC1 expression and function requires systematic analytical approaches:

This systematic approach can reconcile apparently contradictory findings and advance understanding of context-dependent HIC1 functions .