HJURP Antibody

What is HJURP and why is it important in cellular biology?

HJURP functions as a centromeric protein that plays a central role in the incorporation and maintenance of histone H3-like variant CENPA at centromeres. It acts as a specific chaperone for CENPA and is required for the incorporation of newly synthesized CENPA molecules into nucleosomes at replicated centromeres . HJURP prevents CENPA-H4 tetramerization and premature DNA binding by the CENPA-H4 tetramer, and directly binds Holliday junctions .

What are the common applications for HJURP antibodies in research?

HJURP antibodies are utilized in multiple experimental applications:

• Western Blot (WB): For detecting HJURP expression levels in cell and tissue lysates, with an expected band size of around 84 kDa

• Immunohistochemistry (IHC-P): For examining HJURP distribution in formalin-fixed, paraffin-embedded tissue sections

• Immunoprecipitation (IP): For isolating HJURP protein complexes and studying protein-protein interactions

• Immunofluorescence (IF/ICC): For visualizing subcellular localization, particularly at centromeres

• Flow Cytometry (FCM): For analyzing HJURP levels in individual cells

• ELISA: For quantitative detection of HJURP in various sample types

When selecting an HJURP antibody, researchers should verify that the antibody has been validated for their specific application and experimental system.

What technical considerations should be addressed when using HJURP antibodies for immunostaining?

When performing immunostaining with HJURP antibodies, several methodological considerations are important:

• Fixation method: Most validated protocols use 4% paraformaldehyde fixation for immunofluorescence studies of HJURP at centromeres

• Permeabilization: Optimization of permeabilization conditions is critical as excessive treatment may disrupt nuclear architecture

• Antibody dilution: Typically, researchers use HJURP antibodies at 1:100 to 1:1000 dilutions for immunostaining applications

• Cell cycle considerations: HJURP shows cell cycle-dependent localization patterns, with the most prominent centromeric recruitment occurring in early G1 phase

• Controls: Include both positive controls (cell lines known to express HJURP, such as HeLa, U2OS, or MCF-7) and negative controls (antibody diluent only or isotype controls)

For optimal centromeric localization studies, researchers may want to co-stain with established centromere markers (such as CENP-A or CENP-B) to confirm proper localization.

How can researchers validate HJURP antibody specificity for their experimental systems?

Comprehensive validation of HJURP antibodies should include:

• siRNA/shRNA knockdown: Depletion of HJURP should result in decreased signal in Western blot and immunostaining. Studies have shown that efficient HJURP knockdown (to ≤5-10% of control levels) leads to loss of CENP-A at centromeres, which can serve as a functional readout

• Overexpression systems: Expressing tagged versions of HJURP (e.g., GFP-HJURP) and confirming co-localization with the antibody signal

• Multiple antibodies: Using antibodies targeting different HJURP epitopes to confirm consistent results

• Mass spectrometry validation: Confirming that immunoprecipitated proteins include HJURP by mass spectrometry analysis

• Western blot analysis: Verifying a single band of the expected molecular weight (approximately 84 kDa)

For truncation mutant studies, researchers should be aware that different HJURP domains may affect antibody recognition. For example, antibodies targeting the C-terminal region will not detect N-terminal fragments like HJURP 1-482 .

What techniques can be used to study HJURP dimerization, and how can antibodies facilitate this research?

HJURP dimerization is critical for its function, and several approaches can investigate this property:

• Co-immunoprecipitation: Using differentially tagged HJURP constructs (e.g., FLAG and HA) to demonstrate dimerization through co-IP experiments with respective antibodies

• Proximity ligation assay (PLA): Detecting protein-protein interactions in situ using antibodies against endogenous HJURP or tagged versions

• FRET/BRET analysis: Using fluorescently tagged HJURP constructs to measure energy transfer as an indicator of dimerization

• Size exclusion chromatography (SEC): Combined with Western blotting using HJURP antibodies to detect dimeric forms

• Truncation mutant analysis: Research has shown that the HJURP C-terminal region (HJURP 482-end) requires endogenous HJURP for centromeric recruitment, suggesting dimerization

Notably, studies have demonstrated that HJURP dimerization occurs through coiled-coil domains, with the model proposing that two HJURP molecules dimerize to bind two CENP-A/H4 dimers, facilitating tetrasome assembly .

How does HJURP expression correlate with tumor progression and immune infiltration in cancer research?

HJURP has emerged as a potential pan-cancer biomarker with significant implications:

• Expression patterns: HJURP is abnormally expressed in most cancer types and subtypes in the TCGA database, with overexpression generally associated with poor prognosis

• Immune correlation: Research indicates that HJURP expression is significantly related to immunosuppression and tumor-infiltrating immune cells in various cancers, including hepatocellular carcinoma

• Methylation status: HJURP methylation is inversely proportional to mRNA expression levels, which mediates dysfunctional phenotypes of T cells and poor prognosis in different cancer types

• Immune evasion mechanisms: HJURP appears related to tumor immune evasion through different mechanisms, including T cell rejection

For researchers investigating HJURP in cancer contexts, recommended approaches include:

• Multiplex immunohistochemistry with HJURP antibodies alongside immune cell markers

• Flow cytometry analysis of tumor samples to correlate HJURP levels with immune cell populations

• Single-cell RNA sequencing to identify cell clusters expressing abundant HJURP

• Gene Set Enrichment Analysis (GSEA) to link HJURP expression to specific bioprocesses

What are the best experimental approaches to study HJURP's interaction with CENP-A using antibodies?

Investigating HJURP-CENP-A interactions requires specialized techniques:

• Co-immunoprecipitation (Co-IP): Using HJURP antibodies to pull down complexes containing CENP-A, or vice versa

• Chromatin Immunoprecipitation (ChIP): To analyze HJURP localization at centromeric regions

• Biochemical reconstitution: In vitro assembly of HJURP-CENP-A complexes followed by structural and functional analysis

• Microscopy techniques: Super-resolution microscopy with appropriate antibodies to visualize co-localization

• Protein domain mapping: Using HJURP mutants to identify critical interaction regions

Research has demonstrated that HJURP uses distinct CENP-A surfaces for recognition and stabilization. While crystal and NMR structures have provided insights, contradicting data exists on how mammalian HJURP recognizes CENP-A/H4 . For example, mutation of HJURP to remove potential steric clashing with H3 (such as HJURP Trp66) has shown no detectable effect on HJURP recognition in cell-based approaches or in ternary complex formation monitored by SEC .

What are essential controls when using HJURP antibodies in experiments involving siRNA knockdowns?

When conducting HJURP knockdown experiments, proper controls are crucial:

• Scrambled siRNA control: Essential to distinguish between specific and non-specific effects of the siRNA transfection process

• Western blot verification: Confirming knockdown efficiency; studies show effective knockdown typically reduces HJURP to ≤5-10% of control levels

• Multiple siRNA sequences: Using at least two distinct siRNAs (e.g., Si1 and Si2 as described in the literature) targeting different regions of HJURP mRNA to rule out off-target effects

• Rescue experiments: Expressing siRNA-resistant HJURP constructs to confirm phenotype specificity

• Functional readouts: Monitoring CENP-A localization at centromeres as a functional consequence of HJURP depletion

• Time-course analysis: Assessing phenotypes at multiple time points (typically 24-72 hours post-transfection) to capture the full range of effects

Research has demonstrated that HJURP depletion results in not only loss of CENP-A at centromeres but also a reduction in total CENP-A levels to approximately 50% of initial levels, providing a quantifiable metric for knockdown efficiency .

What sample preparation techniques yield optimal results with HJURP antibodies?

Effective sample preparation is critical for reliable HJURP detection:

For Western Blotting:

• Cell lysis buffers containing protease inhibitors are essential to prevent HJURP degradation

• Samples from HeLa, U2OS, and MCF-7 cell lines serve as positive controls for HJURP expression

• Expected band size is approximately 84 kDa, though post-translational modifications may affect migration

• Typically, 2-20 μg of whole cell lysate is sufficient for detection with validated antibodies

For Immunohistochemistry:

• Formalin-fixed, paraffin-embedded (FFPE) tissue sections typically require antigen retrieval, with heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) being commonly effective

• Antibody concentrations around 1 μg/ml have been successfully used for IHC applications

For Immunofluorescence:

• 4% paraformaldehyde fixation for 10-15 minutes at room temperature preserves nuclear architecture

• Permeabilization with 0.2-0.5% Triton X-100 allows antibody access to nuclear proteins

• Cell cycle synchronization may be necessary as HJURP localization varies throughout the cell cycle

How can researchers investigate HJURP's role in the tumor microenvironment using antibodies?

HJURP's emerging role in tumor immunology offers several research approaches:

• Multiplex IHC/IF: Using HJURP antibodies alongside markers for immune cell populations (CD8+ T cells, regulatory T cells, macrophages) to assess spatial relationships

• Flow cytometry: Combining surface immune markers with intracellular HJURP staining to correlate expression with specific cell populations

• Cell sorting: Isolating tumor-infiltrating CD8+ T cells based on PD-1 expression followed by HJURP expression analysis

• Single-cell analysis: Combining HJURP antibodies with other markers to identify cell clusters with distinct expression patterns

• Weighted gene co-expression network analysis (WGCNA): Identifying immunity-related genes whose expression correlates with HJURP

Research has demonstrated that HJURP is significantly related to immunosuppression in hepatocellular carcinoma and other cancer types, suggesting its potential as a biomarker for immunotherapy response .

What techniques can be used to study post-translational modifications of HJURP?

Investigating HJURP post-translational modifications requires specialized approaches:

• Phospho-specific antibodies: For detecting specific phosphorylation sites known to regulate HJURP function

• Immunoprecipitation followed by mass spectrometry: To comprehensively identify modifications

• 2D gel electrophoresis: To separate HJURP isoforms based on charge differences from modifications

• Phos-tag gels: To separate phosphorylated from non-phosphorylated forms of HJURP

• In vitro kinase assays: To identify kinases responsible for HJURP modification

• Cell cycle synchronization: As HJURP modifications likely vary throughout the cell cycle

HJURP has been identified as a 14-3-3-associated AKT substrate (one of its alternative names is FAKTS) , suggesting regulation by the PI3K/AKT pathway. Researchers investigating this aspect should consider inhibitor treatments and phosphorylation-site mutants to elucidate functional significance.

What are common challenges when using HJURP antibodies and how can they be addressed?

Researchers often encounter specific challenges with HJURP antibodies:

How should researchers interpret HJURP expression data in cancer studies?

When analyzing HJURP expression in cancer contexts:

• Expression levels: Multiple studies indicate HJURP is overexpressed in various cancers, correlating with poor prognosis

• Subcellular localization: Changes in HJURP localization patterns may have functional significance beyond mere expression levels

• Correlation with clinical parameters: HJURP expression should be analyzed in relation to tumor stage, grade, metastasis, and patient survival

• Immune correlations: HJURP expression correlates with immune cell infiltration in multiple cancer types, requiring multivariate analysis

• Methylation status: HJURP methylation is inversely proportional to mRNA expression levels and can mediate T cell dysfunction in some cancers

Researchers should adopt a comprehensive approach integrating HJURP expression data with genomic, epigenomic, and clinical information for meaningful interpretation.

What experimental approaches can resolve contradictory findings regarding HJURP function?

Scientific literature contains some contradicting data about HJURP function, particularly regarding:

• CENP-A recognition mechanisms: Despite crystal and NMR structures, contradictory data exists on how mammalian HJURP recognizes CENP-A/H4

• Domain importance: Different studies emphasize various HJURP domains for function

• Cancer correlations: The mechanistic link between HJURP overexpression and cancer progression requires clarification

To resolve these contradictions, researchers should consider:

• Combined methodologies: Integrating structural biology, cell biology, and biochemistry approaches

• Systematic mutagenesis: Creating comprehensive panels of HJURP mutants to map functional domains

• Advanced imaging: Using super-resolution techniques to visualize HJURP-CENP-A interactions in situ

• Multi-omics integration: Combining expression, methylation, and functional data from multiple cancer types

• Standardized reporting: Clearly defining experimental conditions, antibody specifications, and controls used

For example, to address contradictions about HJURP recognition of CENP-A, combining cell-based approaches with biochemical assays like size exclusion chromatography has proven valuable .