HIC1 Antibody

What is HIC1 Antibody and what are its primary research applications?

HIC1 Antibody is a research tool used to detect and study the HIC1 protein, a zinc finger transcriptional repressor that functions as a tumor suppressor. Based on available product information, HIC1 antibodies are primarily used in Western Blot (WB), Immunohistochemistry (IHC), and ELISA applications . These antibodies allow researchers to investigate HIC1's expression patterns, subcellular localization, and functional roles in normal and pathological conditions. For example, the Proteintech HIC1 antibody (24949-1-AP) has been validated for Western Blot at dilutions of 1:500-1:2000 and for IHC at similar dilutions . The antibody's effectiveness has been demonstrated in multiple published studies, particularly in cancer research where HIC1's tumor suppressor role is of significant interest .

Which species reactivity is reported for commonly used HIC1 antibodies?

Commercial HIC1 antibodies have been validated for detecting human and mouse HIC1 proteins, as documented in published research . Specifically, antibodies like Proteintech's 24949-1-AP have demonstrated positive Western Blot detection in human Jurkat cells and mouse NIH/3T3 cells . For immunohistochemistry applications, successful detection has been reported in rat tissues, including lung and stomach specimens . When planning cross-species studies, researchers should carefully verify the antibody's reactivity with their target species, as this validation information is crucial for experimental design and interpretation of results.

What are the recommended storage conditions for HIC1 antibodies?

For optimal performance and longevity, HIC1 antibodies should be stored at -20°C where they typically remain stable for one year after shipment . Many commercial preparations are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . This formulation helps maintain antibody stability and activity. Importantly, aliquoting is generally unnecessary for -20°C storage with these buffer conditions, which simplifies lab management procedures . Some preparations in smaller volumes (e.g., 20μl) may contain 0.1% BSA as a stabilizer . Researchers should follow manufacturer-specific recommendations for their particular antibody to ensure maximum detection sensitivity in experiments.

How can researchers validate the specificity of HIC1 antibodies in their experimental systems?

Validating antibody specificity is crucial for reliable results. For HIC1 antibodies, researchers can employ several complementary approaches:

• Positive and negative control cells/tissues: Use cell lines with known HIC1 expression (e.g., BJ-hTERT immortalized human fibroblasts) as positive controls and compare with HIC1 knockdown versions of the same cells . Published research has demonstrated this approach by showing detection of endogenous HIC1 proteins in BJ-hTERT cells that disappears in BJ-hTERT cells knocked down for HIC1 .

• Overexpression validation: Confirm antibody specificity by detecting HIC1 in cells where it has been experimentally overexpressed (e.g., HEK293T cells transfected with HIC1 expression constructs) .

• siRNA knockdown: Transfect cells with HIC1-specific siRNAs and confirm reduction or absence of signal compared to control siRNA treatment. This approach has been successfully used with WPMY-1 cells, where HIC1-positive nuclear signals disappeared after siRNA treatment .

• Immunofluorescence negative controls: Always include secondary-antibody-only controls in immunofluorescence experiments to rule out non-specific binding .

These validation approaches collectively strengthen confidence in the specificity of HIC1 detection in experimental systems.

What are the characteristic expression patterns of HIC1 observed with antibody detection methods?

When detecting HIC1 using immunofluorescence microscopy, researchers should expect to observe a distinctive subcellular localization pattern:

• Nuclear localization: HIC1 primarily exhibits nuclear localization, consistent with its role as a transcription factor .

• Punctate nuclear structures: Endogenous HIC1 proteins are typically detected in punctate nuclear structures, a pattern characteristic of many BTB/POZ domain-containing proteins . This distinctive pattern can serve as an additional specificity indicator when validating antibody performance.

• Tissue-specific expression patterns: Interestingly, immunohistochemical studies have revealed that in normal prostate and breast tissues, HIC1 expression is predominantly observed in stromal cells rather than epithelial cells . In prostate adenocarcinomas, HIC1 expression was detected in the stromal compartment but not significantly in cancer cells .

Understanding these characteristic expression patterns is essential for correct interpretation of experimental results when studying HIC1 in different cellular and tissue contexts.

What methodological considerations are important when studying HIC1's role in DNA damage response and genomic stability?

When investigating HIC1's functions in maintaining genomic integrity, researchers should consider several methodological approaches:

• Conditional knockout models: Use conditional knockout systems to study acute loss of HIC1 function, as has been done with mouse embryonic fibroblasts (MEFs) . This approach revealed that HIC1 inactivation results in cell cycle arrest, premature senescence, chromosomal instability, and spontaneous transformation .

• Chromosomal stability assays: Employ metaphase spread analysis, micronuclei formation assays, or fluorescence in situ hybridization techniques to assess chromosomal instability following HIC1 modulation.

• DNA damage response pathways: When studying HIC1's role in DNA repair, consider examining its interaction with the ATM-SIRT1-HDAC4 dependent mechanism . Research has shown that HIC1 interacts with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex, which is required for effective DNA repair during S phase .

• Replication stress models: Since HIC1 appears to maintain genome integrity during sustained replicative stress, consider using replication stress-inducing agents (e.g., hydroxyurea, aphidicolin) in experimental designs to better understand this function .

• Cooperative oncogenic models: To study HIC1's tumor suppressor function in vivo, consider models that combine HIC1 inactivation with oncogene activation, such as the conditional KRasG12D lung adenocarcinoma model that demonstrates cooperation between oncogenic KRas and loss of HIC1 .

How does HIC1 expression differ between normal and cancerous tissues as detected by antibodies?

Understanding HIC1's differential expression patterns is crucial for cancer research applications. Based on immunohistochemical studies:

• Normal tissues: In normal prostate and breast tissues, HIC1 protein expression was not detected in epithelial cells but rather in stromal cells, appearing as strong nuclear staining consistent with its function as a transcription factor .

• Cancer tissues: In prostate adenocarcinomas, HIC1 expression was similarly not detected in cancer cells but maintained in the stromal compartment . This pattern of expression challenges conventional understanding of HIC1 as a tumor suppressor primarily functioning in epithelial cells.

• Epigenetic silencing: In heterozygous HIC1 knockout mice that develop tumors, the remaining wild-type HIC1 allele undergoes epigenetic silencing marked by DNA methylation . This suggests that antibody-based detection methods should be complemented with epigenetic analyses when studying HIC1 in cancer contexts.

These findings highlight the importance of examining both epithelial and stromal compartments when investigating HIC1 expression in cancer tissues, and the need to correlate protein expression data with epigenetic status of the HIC1 locus.

What antigen retrieval methods are recommended for optimal HIC1 detection in tissue samples?

For successful immunohistochemical detection of HIC1 in tissue samples, researchers should consider the following antigen retrieval approaches:

• Primary recommendation: Use TE buffer at pH 9.0 for antigen retrieval in tissues such as rat lung and stomach .

• Alternative approach: Citrate buffer at pH 6.0 can be used as an alternative antigen retrieval method .

The choice between these methods may depend on tissue type, fixation protocols, and the specific antibody being used. Researchers should optimize conditions for their particular experimental system, as antigen retrieval can significantly impact the sensitivity and specificity of HIC1 detection in fixed tissues.

How can researchers effectively study HIC1's tumor suppressor function in experimental models?

Based on published research findings, several approaches can be employed to investigate HIC1's tumor suppressor functions:

• Heterozygous knockout mouse models: HIC1 heterozygous mice develop various tumors beginning after 70 weeks, with 16.4% developing tumors by 90 weeks compared to 0% in wild-type littermates . This model is valuable for studying the long-term consequences of HIC1 haploinsufficiency.

• Sex-specific tumor development: Researchers should consider sex as a biological variable, as male HIC1+/- mice develop predominantly epithelial cancers while females exhibit a preponderance of sarcomas and lymphomas . This sex-determined pattern provides insights into tissue-specific roles of HIC1.

• Combination with oncogene activation: The cooperation between HIC1 loss and oncogene activation can be studied using models such as the conditional KRasG12D lung adenocarcinoma model . This approach demonstrates how HIC1 loss contributes to malignant transformation in the context of oncogenic drivers.

• Epigenetic silencing assessment: When using these models, researchers should monitor the epigenetic status of the remaining wild-type HIC1 allele, as epigenetic silencing through DNA methylation is a common second hit in tumors from heterozygous animals .

• Pathway interactions: Consider investigating HIC1's interactions with other tumor suppressor pathways, including p53 and Rb. HIC1 is linked to the Rb pathway, and when Rb is phosphorylated, it releases E2F1, whose target genes mediate various cellular processes .

What experimental approaches can be used to study the relationship between HIC1 and chromosomal stability?

Research has revealed an unexpected function of HIC1 in maintaining chromosomal stability. To investigate this aspect, researchers can employ these methodological approaches:

• Conditional knockout systems: Use conditional HIC1 knockout in primary cells (such as MEFs) to study chromosomal stability after acute loss of HIC1 function . This approach demonstrated that HIC1 inactivation results in chromosomal instability phenocopying the effects of deleting Brca1 .

• DNA damage response mechanisms: Examine HIC1's role in DNA double-strand break repair through interaction with the ATM-SIRT1-HDAC4 pathway . Specifically, investigate HIC1's interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex required for effective DNA repair during S phase .

• Replicative stress models: Since HIC1 appears to maintain genome integrity during sustained replicative stress, designing experiments that induce replication stress can help elucidate this function .

• Transcriptional response to DNA damage: Investigate HIC1's role in mounting a transcriptional response to DNA double-strand breaks, as some research suggests this may be more significant than direct promotion of DNA repair .

These approaches collectively provide a comprehensive experimental framework for understanding HIC1's role in maintaining genomic integrity, which appears to be independent of its conventional role as a transcriptional repressor targeting genes like SIRT1.

What are the recommended experimental controls when using HIC1 antibody in research?

To ensure reliable and interpretable results when using HIC1 antibodies, researchers should incorporate these essential controls:

Additionally, researchers should consider tissue-specific controls, especially given HIC1's differential expression between epithelial and stromal compartments . When studying HIC1 in cancer contexts, including normal adjacent tissue is highly recommended due to the frequent epigenetic silencing of HIC1 in tumors .

What are the optimal antibody dilutions and detection protocols for different HIC1 applications?

Based on published research and product information, researchers should consider these application-specific recommendations:

Key protocol considerations:

• Western Blot: HIC1 is typically observed at 65-70 kDa despite a calculated weight of 75 kDa .

• IHC: Pay attention to both epithelial and stromal compartments, as HIC1 expression is predominantly stromal in some tissues .

• Tissue-specific optimization: Different tissues may require protocol optimization for optimal signal-to-noise ratio .

• Multiple detection methods: When possible, validate findings using multiple detection techniques (e.g., IF, IHC, and WB) as done in published research .

These recommendations should be optimized for each research context, with antibody titration performed to determine the ideal working concentration for specific experimental systems.

What are emerging applications of HIC1 antibodies in cancer research and beyond?

Based on current research findings, several promising directions for HIC1 antibody applications are emerging:

• Biomarker development: Given HIC1's frequent epigenetic silencing in cancers, antibody-based detection of HIC1 protein could complement DNA methylation studies to develop comprehensive biomarker panels for cancer diagnosis and prognosis .

• Tumor-stroma interactions: The unexpected finding that HIC1 is predominantly expressed in stromal rather than epithelial cells in some tissues opens new avenues for investigating tumor-stroma interactions mediated by HIC1 .

• Chromosomal instability mechanisms: HIC1 antibodies can be utilized to further characterize the unexpected role of HIC1 in maintaining chromosomal stability, particularly in the context of DNA repair mechanisms during replication stress .

• Therapeutic response prediction: Since HIC1 status affects genomic stability, antibody-based HIC1 detection might help predict tumor responses to DNA damaging therapies or PARP inhibitors .

• Developmental studies: Given the embryonic lethality of homozygous HIC1 deletion, antibodies could help map HIC1 expression during development to better understand its non-cancer related functions .

These emerging applications highlight the continuing importance of HIC1 antibodies as key research tools for uncovering novel aspects of HIC1 biology in both normal and pathological contexts.