HES2 Antibody

What is the HES2 protein and what are its key functions in cellular processes?

HES2 (Hairy and Enhancer of Split 2) is a transcriptional repressor belonging to the basic helix-loop-helix (bHLH) transcription factor family. It plays a crucial role in gene expression regulation during embryonic development, particularly within the Notch signaling pathway, which is essential for cell differentiation and tissue patterning . The protein forms complexes with TLE proteins involved in transcriptional repression, with this interaction mediated by the carboxy-terminal WRPW motif, enabling effective inhibition of transcriptional activator activity . This regulatory mechanism maintains balance between cell proliferation and differentiation, particularly important in neural and muscle development.

HES2 contains a specialized basic domain with a helix-interrupting proline that preferentially binds to N-box (CACNAG) sequences rather than canonical E-box (CANNTG) motifs typically recognized by other bHLH proteins . The protein's C-terminal WRPW motif functions as a transcriptional repression domain necessary for interaction with corepressors of the Groucho/TLE family . HES2 is expressed in various embryonic and adult tissues, with notable expression in placenta and several cancer types including pancreatic cancer, colon cancer with RER, cervical cancer, and head and neck tumors .

What types of HES2 antibodies are commercially available and what are their applications?

Multiple types of HES2 antibodies are available for research applications, each with specific characteristics suitable for different experimental approaches:

• Monoclonal Antibodies:

  • Mouse monoclonal IgG1 kappa light chain antibody (clone H-8) detects HES2 protein from mouse, rat, and human samples

  • Mouse monoclonal antibody (clone 1D5, IgG2bκ isotype) specific for human HES2

• Polyclonal Antibodies:

  • Rabbit polyclonal IgG antibodies with reactivity to human, mouse, rat, and primate HES2

These antibodies are validated for multiple applications including western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC) . Both unconjugated forms and various conjugated versions (including agarose, HRP, PE, FITC, and Alexa Fluor® conjugates) are available for specialized applications .

What are the optimal storage conditions for maintaining HES2 antibody activity?

Proper storage is critical for maintaining antibody activity and preventing degradation. Based on manufacturer recommendations, the following storage protocols should be implemented:

• Short-term storage: Store at 4°C (refrigeration)

• Long-term storage: Store at -20°C, with antibodies aliquoted to minimize freeze-thaw cycles

• Shipping conditions: Most HES2 antibodies are shipped with ice packs and should be stored immediately upon receipt at the recommended temperature

• Formulation considerations: Many HES2 antibodies are supplied in buffers containing stabilizers such as PBS with 50% glycerol and 0.02% sodium azide or other protein protectants

It is crucial to avoid repeated freeze-thaw cycles as these significantly reduce antibody activity . For optimal results, smaller working aliquots should be prepared upon receipt to minimize the need for repeated freezing and thawing of the entire stock.

How should I determine the appropriate dilution for HES2 antibody in my experiments?

Optimal dilution ratios vary based on both the specific antibody and the application. The following dilution ranges are recommended based on manufacturer guidelines:

For optimal results, researchers should perform a titration experiment using different dilutions of antibody on control samples with known expression levels of HES2. Start with the manufacturer's recommended range and adjust based on signal-to-noise ratio. When switching to a new batch of the same antibody, validation of the optimal dilution should be repeated to account for potential lot-to-lot variations in antibody concentration and affinity.

What methodological considerations should be taken into account when using HES2 antibodies for detecting low-abundance protein expression?

When studying HES2 in contexts with low protein expression, several methodological refinements can enhance detection sensitivity:

• Signal Amplification Systems:

  • Consider using tyramide signal amplification (TSA) for immunohistochemistry applications

  • For Western blotting, enhanced chemiluminescence (ECL) substrates with higher sensitivity can improve detection limits

  • Utilize biotin-streptavidin amplification systems with HRP-conjugated secondary antibodies

• Sample Preparation Optimization:

  • Enrich for nuclear fractions, as HES2 is primarily localized in the nucleus

  • Use proteasome inhibitors (e.g., MG132) during sample preparation to prevent rapid degradation of HES2

  • Optimize protein extraction buffers with appropriate detergents (0.1-1% NP-40 or Triton X-100) to effectively solubilize nuclear proteins

• Detection Systems:

  • For immunofluorescence, consider using conjugated primary antibodies (such as HES2 Antibody with Alexa Fluor® conjugates)

  • Implement fluorescent secondary antibodies with longer wavelengths to reduce background autofluorescence

  • Use high-sensitivity imaging systems with cooled CCD cameras for fluorescence applications

• Controls:

  • Include positive controls from tissues known to express HES2 (placenta, specific cancer cell lines)

  • Implement peptide competition assays to verify antibody specificity

  • Consider using HES2 overexpression systems as positive controls

The observed molecular weight for HES2 in Western blot applications is approximately 19 kDa , although the theoretical molecular weight is 33.77 kDa , which should be noted when interpreting results.

How can I optimize immunohistochemistry protocols for detecting HES2 in formalin-fixed, paraffin-embedded tissues?

Optimizing IHC protocols for FFPE tissues requires attention to several critical factors:

• Antigen Retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) at 95-98°C for 20-30 minutes

  • Enzymatic retrieval using proteinase K may be considered for certain tissues, but this should be empirically determined

  • Pressure cooker or microwave-based retrieval systems often provide more consistent results than water bath methods

• Blocking Parameters:

  • Implement dual blocking approach: first block endogenous peroxidase activity (3% H₂O₂ for 10 minutes), then block non-specific binding (5% normal serum from secondary antibody species)

  • Consider using commercial blocking reagents containing both protein blockers and detergents

  • For tissues with high endogenous biotin, use avidin-biotin blocking kits prior to antibody incubation

• Antibody Incubation:

  • For rabbit polyclonal HES2 antibodies, dilution ranges of 1:100-1:400 are recommended

  • Overnight incubation at 4°C often yields better results than shorter incubations at room temperature

  • Use humidity chambers to prevent section drying during incubation

• Detection Systems:

  • Polymer-based detection systems often provide superior results compared to traditional ABC methods

  • Consider using amplification systems for tissues with low HES2 expression

  • Optimize DAB development time through careful monitoring to achieve optimal signal-to-noise ratio

• Validated Controls:

  • Include tissues with known HES2 expression: rat heart, rat brain, and rat testis have been validated for IHC applications

  • Include negative controls by omitting primary antibody or using isotype control antibodies

What strategies can be employed to verify the specificity of HES2 antibody signals in experimental systems?

Verifying antibody specificity is crucial for ensuring reliable research outcomes. Multiple complementary approaches should be implemented:

• Genetic Knockdown/Knockout Validation:

  • Use siRNA or CRISPR-Cas9 to reduce or eliminate HES2 expression, then confirm corresponding reduction in antibody signal

  • Employ inducible expression systems to demonstrate signal correlation with controlled HES2 expression levels

• Peptide Competition Assays:

  • Pre-incubate the HES2 antibody with excess immunizing peptide before application

  • For polyclonal antibodies raised against specific peptides (such as the 8-25 amino acid region or 40-120 amino acid range ), use the corresponding synthetic peptides

  • A significant reduction in signal indicates specific binding to the target epitope

• Multiple Antibody Validation:

  • Compare results using different HES2 antibodies targeting distinct epitopes

  • Use both monoclonal (clone H-8 or clone 1D5 ) and polyclonal antibodies for cross-validation

  • Consistent detection patterns across different antibodies support specificity

• Heterologous Expression Systems:

  • Express tagged versions of HES2 in cell lines with low endogenous expression

  • Demonstrate co-localization of tag-specific antibody signals with HES2 antibody signals

  • Show absence of signal in empty vector transfected controls

• Mass Spectrometry Validation:

  • Perform immunoprecipitation using the HES2 antibody followed by mass spectrometry analysis

  • Confirm that peptides identified correspond to HES2 protein

  • Analyze non-specific binding partners to assess potential cross-reactivity

How can I investigate HES2 protein interactions and complex formation using immunoprecipitation approaches?

HES2 functions through interactions with various proteins, particularly TLE family corepressors. The following IP strategy can effectively capture these interactions:

• Nuclear Extract Preparation:

  • Since HES2 is predominantly nuclear-localized , optimize nuclear extraction protocols

  • Use gentle lysis buffers (e.g., 20 mM HEPES pH 7.9, 150 mM NaCl, 0.5% NP-40, 10% glycerol) supplemented with protease and phosphatase inhibitors

  • Include 1-2 mM DTT to preserve protein-protein interactions involving cysteine residues

• Immunoprecipitation Protocol:

  • Pre-clear nuclear extracts with protein A/G beads to reduce non-specific binding

  • Use 2-5 μg of HES2 antibody per 500 μg of nuclear extract

  • The mouse monoclonal HES2 antibody (H-8) has been validated for immunoprecipitation applications

  • Consider using agarose-conjugated HES2 antibody (HES2 Antibody H-8 AC) for direct IP without secondary antibody beads

• Co-Immunoprecipitation for Binding Partners:

  • Focus on known interaction partners such as TLE proteins that bind via the C-terminal WRPW motif

  • Include appropriate detergent conditions (0.1-0.5% NP-40) to maintain interactions while reducing background

  • After IP, perform Western blotting for both HES2 (to confirm successful precipitation) and potential binding partners

• Controls and Validation:

  • Include IgG control immunoprecipitations to establish background binding levels

  • For TLE interaction studies, consider using wild-type HES2 versus WRPW motif mutants to demonstrate specificity

  • Validate interactions through reciprocal co-IPs (i.e., IP with anti-TLE antibody and blot for HES2)

• Analysis of Post-Translational Modifications:

  • After IP, probe for potential modifications (phosphorylation, acetylation, ubiquitination) that may regulate HES2 function

  • Consider using modification-specific antibodies in Western blotting after HES2 immunoprecipitation

What approaches can be used to investigate HES2 DNA binding specificity and genomic targets?

HES2 binds to specific DNA sequences as part of its transcriptional regulatory function. The following methods can characterize its DNA binding properties and genomic targets:

• Chromatin Immunoprecipitation (ChIP):

  • Use cross-linking agents (1% formaldehyde for 10 minutes) to stabilize DNA-protein interactions

  • Sonicate chromatin to 200-500 bp fragments for optimal resolution

  • Immunoprecipitate using 3-5 μg of HES2 antibody per ChIP reaction

  • Include appropriate controls (IgG ChIP, input samples)

  • Analyze enriched regions by qPCR (for known targets) or sequencing (for genome-wide analysis)

• DNA Binding Specificity Assays:

  • Electrophoretic Mobility Shift Assays (EMSA) using recombinant HES2 or nuclear extracts

  • Focus on N-box (CACNAG) sequences, which are preferentially bound by HES2's specialized basic domain

  • Compare binding to canonical E-box (CANNTG) sequences to demonstrate binding specificity

  • Use competition assays with unlabeled probes to confirm sequence-specific binding

• Reporter Gene Assays:

  • Construct luciferase reporters containing predicted HES2 binding sites

  • Test HES2-mediated repression through co-transfection experiments

  • Use site-directed mutagenesis of N-box sequences to confirm specificity

  • Test the functional importance of the WRPW motif by comparing wild-type and mutant HES2 constructs

• Genome-wide Approaches:

  • ChIP-seq to identify all genomic binding sites

  • RNA-seq following HES2 knockdown/overexpression to identify genes with expression changes

  • Integration of ChIP-seq and RNA-seq data to identify direct transcriptional targets

  • Motif enrichment analysis to confirm binding site preferences in vivo

What are common causes of non-specific background in Western blots using HES2 antibodies and how can they be addressed?

Non-specific background in Western blotting can significantly complicate data interpretation. Several strategies can minimize these issues:

• Antibody-Related Factors:

  • Optimize primary antibody dilution: for Western blotting, use 1:500-1:2000 for polyclonal antibodies or 1-5 μg/mL for monoclonal antibodies

  • Extend washing steps (4-5 times, 5-10 minutes each) with TBST (TBS + 0.1% Tween-20)

  • Consider using more specific monoclonal antibodies (such as clone H-8 or clone 1D5 ) rather than polyclonal antibodies if background persists

  • Pre-adsorb polyclonal antibodies with cell/tissue lysates from HES2-negative samples

• Blocking Optimization:

  • Test different blocking agents (5% non-fat dry milk, 5% BSA, or commercial blocking buffers)

  • Extend blocking time to 1-2 hours at room temperature or overnight at 4°C

  • Add 0.1-0.2% Tween-20 to blocking buffer to reduce hydrophobic interactions

• Sample Preparation Improvements:

  • Ensure complete protein denaturation by heating samples at 95°C for 5 minutes in loading buffer

  • Include reducing agents (DTT or β-mercaptoethanol) in sample buffer

  • Centrifuge samples after heating to remove insoluble material

  • Consider nuclear extraction protocols to enrich for HES2, which is primarily nuclear-localized

• Membrane Handling:

  • Use PVDF membranes instead of nitrocellulose for potentially better signal-to-noise ratio

  • After transfer, rinse membranes thoroughly in TBST before blocking

  • Consider membrane-specific blocking agents recommended by membrane manufacturers

How can I optimize immunofluorescence protocols to achieve high-quality HES2 localization data?

Achieving clear immunofluorescence results requires attention to fixation, permeabilization, and detection parameters:

• Fixation and Permeabilization:

  • For nuclear proteins like HES2, 4% paraformaldehyde fixation (15-20 minutes) followed by 0.1-0.5% Triton X-100 permeabilization (10 minutes) is typically effective

  • Test different fixatives (methanol, acetone, or PFA) if initial results are suboptimal

  • For certain applications, methanol fixation (-20°C, 10 minutes) may provide better nuclear antigen accessibility

• Blocking and Antibody Incubation:

  • Block with 5-10% normal serum (from secondary antibody species) with 0.1-0.3% Triton X-100

  • Consider adding 1-2% BSA to reduce non-specific binding

  • For primary antibody incubation, dilute appropriately and incubate overnight at 4°C in a humidified chamber

  • For HES2 antibody (H-8), which has been validated for immunofluorescence applications , follow manufacturer's recommended dilutions

• Signal Detection:

  • Use directly conjugated primary antibodies (such as HES2 antibody with fluorophore conjugates) to reduce background

  • When using secondary antibodies, highly cross-adsorbed versions minimize species cross-reactivity

  • Include DAPI or other nuclear counterstains to visualize nuclear localization of HES2

  • Mount slides using anti-fade mounting media to prevent photobleaching

• Controls and Validation:

  • Include negative controls (primary antibody omission, isotype controls)

  • Use positive control cells/tissues with known HES2 expression

  • Consider siRNA knockdown controls to demonstrate specificity

  • Perform z-stack imaging to accurately determine subcellular localization

How can I quantitatively analyze HES2 expression in different experimental conditions?

Quantitative analysis of HES2 requires careful attention to methodology and normalization:

• Western Blot Quantification:

  • Use gradient gels (4-20%) for optimal separation and resolution of the 19 kDa HES2 band

  • Load equal amounts of protein (validated by BCA or Bradford assay)

  • Include loading controls appropriate for nuclear proteins (e.g., Lamin B1, Histone H3)

  • Use fluorescent secondary antibodies or digital chemiluminescence systems for wider linear dynamic range

  • Analyze band intensities using software such as ImageJ, normalizing to loading controls

• qPCR Analysis of HES2 mRNA Levels:

  • Design gene-specific primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Validate primer efficiency using standard curves

  • Use multiple reference genes (GAPDH, ACTB, 18S rRNA) for normalization

  • Analyze data using the 2^(-ΔΔCt) method or absolute quantification with standard curves

• Immunohistochemistry Quantification:

  • Use digital image analysis software to quantify staining intensity

  • Develop scoring systems based on both staining intensity and percentage of positive cells

  • Implement automated tissue analysis platforms for unbiased assessment

  • Include calibration standards in each staining batch to account for batch-to-batch variation

• Flow Cytometry for Single-Cell Analysis:

  • Optimize fixation and permeabilization protocols for nuclear antigens

  • Use fluorophore-conjugated HES2 antibodies for direct detection

  • Include appropriate isotype controls to establish background levels

  • Analyze both frequency of positive cells and mean fluorescence intensity

How can HES2 antibodies be utilized to investigate the role of Notch signaling in cancer progression?

HES2 expression has been documented in various cancer types, including pancreatic cancer, colon cancer with RER, cervical cancer, and head and neck tumors . The following methodological approaches can help elucidate its role in cancer:

• Tissue Microarray Analysis:

  • Use HES2 antibodies for IHC on cancer tissue microarrays

  • Recommended dilutions for IHC applications range from 1:100-1:400

  • Correlate HES2 expression with clinical parameters (stage, grade, patient outcome)

  • Perform multiplexed IHC to simultaneously detect HES2 and other Notch pathway components

• Cell Line Models:

  • Screen cancer cell line panels for HES2 expression using Western blotting (1:500-1:2000 dilution)

  • Generate stable HES2 knockdown or overexpression cell lines

  • Assess effects on proliferation, migration, invasion, and therapy resistance

  • Investigate synergy with Notch pathway inhibitors (γ-secretase inhibitors, anti-Notch antibodies)

• Xenograft Models:

  • Develop in vivo models with modulated HES2 expression

  • Use HES2 antibodies for IHC analysis of tumor sections

  • Correlate HES2 expression with tumor growth, angiogenesis, and metastatic potential

  • Assess effects of Notch pathway modulation on HES2 expression in vivo

• Patient-Derived Models:

  • Analyze HES2 expression in patient-derived xenografts and organoids

  • Correlate expression with treatment response

  • Implement HES2 as a potential biomarker for patient stratification in clinical trials

What methodological approaches can be used to study interactions between HES2 and other transcriptional regulators in developmental contexts?

HES2's role in development involves complex interactions with various transcriptional regulators:

• Co-Immunoprecipitation Studies:

  • Use HES2 antibodies validated for IP applications to pull down protein complexes

  • Focus on known interactors like TLE proteins that bind via the WRPW motif

  • Analyze samples from different developmental stages to track temporal dynamics of interactions

  • Implement tandem affinity purification for higher purity complex isolation

• Proximity Ligation Assays (PLA):

  • Visualize in situ protein-protein interactions at single-molecule resolution

  • Combine HES2 antibodies with antibodies against potential interaction partners

  • Analyze spatial and temporal patterns of interactions during development

  • Quantify interaction frequencies in different cell types or developmental stages

• ChIP-reChIP (Sequential ChIP):

  • Identify genomic loci co-occupied by HES2 and other transcription factors

  • First immunoprecipitate with HES2 antibody, then re-immunoprecipitate with antibody against potential partner

  • Compare binding profiles at different developmental timepoints

  • Integrate with gene expression data to identify cooperatively regulated targets

• CRISPR-Based Approaches:

  • Generate tagged endogenous HES2 for more physiological interaction studies

  • Implement domain-specific mutations to disrupt specific interactions

  • Use CUT&RUN or CUT&Tag methods with HES2 antibodies for higher resolution chromatin binding profiles

  • Combine with single-cell approaches to address cellular heterogeneity

How can advanced imaging techniques be combined with HES2 antibodies to study its dynamic regulation in live cells?

Emerging imaging technologies offer new opportunities for studying HES2 dynamics:

• Live-Cell Imaging Approaches:

  • Use fluorescently tagged HES2 constructs to track protein dynamics

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to analyze protein mobility

  • Apply FLIM-FRET (Fluorescence Lifetime Imaging Microscopy - Förster Resonance Energy Transfer) to study protein-protein interactions in real-time

  • Correlate with fixed-cell immunofluorescence using HES2 antibodies for validation

• Super-Resolution Microscopy:

  • Apply techniques like STORM, PALM, or SIM for nanoscale localization of HES2

  • Use HES2 antibodies with appropriate fluorophores optimized for super-resolution imaging

  • Investigate subnuclear localization and potential association with specific nuclear domains

  • Combine with proximity labeling approaches to identify proteins in the immediate vicinity

• Correlative Light and Electron Microscopy (CLEM):

  • Use gold-conjugated secondary antibodies to visualize HES2 localization at ultrastructural level

  • Correlate immunofluorescence patterns with electron microscopy data

  • Investigate association with specific nuclear substructures at nanometer resolution

• Expansion Microscopy:

  • Apply physical expansion of samples to achieve super-resolution with standard microscopes

  • Optimize HES2 antibody immunostaining protocols for expanded samples

  • Visualize fine details of nuclear organization and HES2 distribution

What methodological considerations are important when designing CRISPR-based genomic editing experiments to study HES2 function?

CRISPR-based approaches offer powerful tools for HES2 functional studies:

• Guide RNA Design:

  • Target functional domains (basic domain, HLH domain, Orange domain, WRPW motif) for domain-specific studies

  • Use multiple guide RNAs to increase editing efficiency

  • Consider potential off-target effects using prediction algorithms

  • Design repair templates for precise gene editing or tagging

• Validation Strategies:

  • Use HES2 antibodies to confirm protein knockout or modification

  • For Western blotting, use antibodies at 1:500-1:2000 dilution (polyclonal) or 1-5 μg/mL (monoclonal)

  • Sequence edit sites to confirm intended modifications

  • Perform off-target analysis at predicted sites

• Functional Readouts:

  • Analyze expression of known HES2 target genes

  • Assess developmental phenotypes in appropriate model systems

  • Investigate effects on Notch pathway activity

  • Examine cell differentiation dynamics, particularly in neural and muscle lineages

• Advanced CRISPR Applications:

  • Implement CRISPRi/CRISPRa for reversible modulation of HES2 expression

  • Use CRISPR base editors for precise mutation introduction without double-strand breaks

  • Apply CRISPR screening approaches to identify genetic interactors

  • Generate cell/animal models with fluorescently tagged endogenous HES2 for live imaging