HES7 Antibody, HRP conjugated

What is HES7 and why is it significant in research?

HES7 is a bHLH (basic helix-loop-helix) transcription factor that functions as a transcriptional repressor. It plays a critical role in somitogenesis - the process of somite formation in vertebrate embryos. Research has established that HES7 displays cyclic expression of mRNA in the presomitic mesoderm (PSM), and the protein is also expressed in a dynamic manner . This periodic expression is essential for proper segmentation during embryonic development, making HES7 a key target in developmental biology research .

HES7 functions through a negative feedback loop, where it represses its own transcription as well as that of other genes like Lunatic fringe (Lfng) . This auto-regulatory mechanism represents a molecular basis for the segmentation clock that controls somite formation. Mutations in HES7 are associated with spondylocostal dysostosis type 4 (SCDO4), a rare condition characterized by vertebral and costal anomalies .

What is HRP conjugation and what advantages does it offer?

Horseradish peroxidase (HRP) is a 44 kDa glycoprotein containing 18% carbohydrate content surrounding a protein core . When conjugated to antibodies, HRP serves as a reporter molecule that enables visualization through enzymatic reactions. The conjugation process typically involves creating a stable, covalent linkage between the enzyme and the antibody without affecting the antigen-binding capability of the antibody or the catalytic activity of the enzyme .

Advantages of HRP-conjugated antibodies include:

• High sensitivity in detection systems

• Stability (can be stored at 4°C for up to 6 months)

• Compatibility with multiple detection methods (chromogenic, chemiluminescent)

• Small size allowing better tissue penetration compared to other enzyme labels

• Cost-effectiveness compared to other detection systems

• Low endogenous background in most mammalian tissues

What are the typical applications for HES7 antibody-HRP conjugates?

HES7 antibody-HRP conjugates are primarily utilized in:

• Western Blotting: For detecting HES7 protein expression in tissue or cell lysates. This is particularly useful for studying the dynamic expression patterns of HES7 in different developmental stages or experimental conditions .

• Immunohistochemistry (IHC): For visualizing the spatial distribution of HES7 protein in tissue sections. This is crucial for understanding the localization patterns in presomitic mesoderm and other tissues .

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative measurement of HES7 protein levels in solution samples .

• Chromatin Immunoprecipitation (ChIP): For studying the interaction between HES7 and its target DNA sequences, which is essential for understanding its role as a transcriptional regulator .

How should researchers select the appropriate HES7 antibody for HRP conjugation?

Selection criteria should include:

• Specificity: Verify that the antibody recognizes HES7 without cross-reactivity to other HES family members. This is particularly important given the sequence homology between different HES proteins.

• Species reactivity: Consider the experimental model system. Available HES7 antibodies show different species reactivity profiles:

  • Human-specific HES7 antibodies

  • Multi-species reactive antibodies (Human, Mouse, Rat, etc.)

• Application validation: Confirm that the antibody has been validated for the intended application. For example, antibody ABIN6735843 is validated for WB, IHC, and IHC (p), while ABIN7245221 is validated for ELISA and IHC .

• Epitope location: Consider the target region of the antibody. Some HES7 antibodies target the N-terminal region (amino acids 33-63) , while others may target internal regions . This is particularly important when studying specific domains of HES7.

• Buffer compatibility: Ensure the antibody buffer is compatible with the conjugation chemistry. Common buffer additives can hamper the conjugation process .

What are the optimal methods for HRP conjugation to HES7 antibodies?

Based on the literature, two primary methods are recommended:

1. Periodate Method (Enhanced by Lyophilization):

This method is particularly effective for obtaining high-sensitivity conjugates:

• Step 1: Activate HRP using 0.15 M sodium metaperiodate to generate aldehyde groups by oxidizing carbohydrate moieties

• Step 2: Dialyze activated HRP against 1× PBS for 3 hours at room temperature

• Step 3: Freeze activated HRP at -80°C for 5-6 hours

• Step 4: Lyophilize the frozen HRP overnight

• Step 5: Mix lyophilized HRP with purified anti-HES7 antibody (1 mg/ml) in a 4:1 molar ratio (HRP:antibody)

• Step 6: Incubate at 37°C for 1 hour

• Step 7: Add 1/10th volume of sodium cyanoborohydride for Schiff's base formation

• Step 8: Incubate at 4°C for 2 hours

• Step 9: Perform overnight dialysis against 1× PBS at room temperature

• Step 10: Add stabilizers and store at 4°C for up to 6 months or at -20°C for long-term storage

This enhanced method allows for storage of active HRP at 4°C for longer duration and increases sensitivity due to higher conjugation efficiency .

2. Lightning-Link® HRP Method:

A commercial approach that simplifies the conjugation process:

• Ensures direct conjugation of antibody to HRP without additional laborious steps

• Particularly useful for time-sensitive protocols

• Eliminates the need for traditional, labor-intensive methodologies

How should researchers optimize HES7-HRP antibody dilutions for different applications?

For optimal results, researchers should:

• Always perform a dilution series during optimization

• Consider signal-to-noise ratio rather than absolute signal intensity

• Include positive and negative controls for each experiment

• Test multiple incubation times (1 hour, 2 hours, overnight) with different dilutions

How can researchers effectively study the dynamic expression of HES7 using HRP-conjugated antibodies?

Given HES7's cyclic expression pattern in the presomitic mesoderm (PSM), specialized approaches are needed:

• Temporal profiling: To capture the dynamic expression of HES7 protein, researchers should consider:

  • Collecting samples at multiple time points spanning the oscillation period

  • Using whole-mount immunostaining with anti-HES7-HRP antibodies for visualization of protein expression patterns

  • Implementing dual labeling with markers for spatial alignment (e.g., Uncx4.1)

• Spatial-temporal correlation: As demonstrated in the literature, the expression domains of HES7 protein and mRNA are overlapping but distinct. Researchers should:

  • Perform parallel detection of HES7 protein (using HRP-conjugated antibodies) and active transcription (using intron probes for nascent transcripts)

  • Bisect PSM tissues and process adjacent sections for protein and mRNA detection

• Protein degradation kinetics: Since HES7 protein undergoes rapid degradation via the ubiquitin-proteasome system:

  • Consider using proteasome inhibitors (e.g., MG132) in control experiments to confirm degradation pathways

  • Design time-course experiments that account for the short half-life of HES7 protein

What controls are essential when using HES7 antibody-HRP conjugates?

A comprehensive control strategy should include:

• Negative controls:

  • Tissue/cells from HES7 knockout models (HES7-null mice show no detectable immunoreactivity)

  • Incubation with isotype-matched irrelevant antibodies conjugated to HRP

  • Omission of primary antibody while maintaining secondary detection system

• Positive controls:

  • Tissues with known HES7 expression (e.g., presomitic mesoderm of E9.5 mouse embryos)

  • Recombinant HES7 protein spiked into negative samples

• Specificity controls:

  • Pre-absorption of antibody with immunizing peptide

  • Testing in tissues expressing related HES family proteins to confirm specificity

  • Western blot analysis confirming single band of appropriate molecular weight

• Methodology controls for conjugation:

  • Unconjugated HRP to assess non-specific binding

  • Unconjugated antibody followed by anti-species-HRP to compare direct vs. indirect detection sensitivity

How can researchers optimize chromatin immunoprecipitation (ChIP) using HES7-HRP antibodies?

For studying HES7's interactions with target promoters (e.g., HES7 and Lfng promoters), consider these optimizations:

• Cross-linking conditions:

  • Use fresh formaldehyde (1% final concentration) for 10 minutes at room temperature

  • Quench with glycine (125 mM final concentration)

• Chromatin preparation:

  • Isolate PSM tissues specifically, as HES7 expression is restricted to this region

  • Optimize sonication to obtain chromatin fragments of 200-500 bp

• Immunoprecipitation:

  • Use purified anti-HES7 antibodies rather than direct HRP conjugates for the pull-down

  • Include negative control with preimmune serum

• Target validation:

  • Design primers that span the putative binding sites in the HES7 promoter:

    • RBP-J binding sites

    • E-boxes and N-boxes (target sequences for HES7 protein)

  • Assess enrichment at the Lfng promoter region, another demonstrated target of HES7

• Analysis:

  • Quantify enrichment by qPCR relative to input and IgG control

  • Consider sequencing ChIP products to identify genome-wide binding sites

What are the considerations for detecting both HES7 protein and transcriptional activity simultaneously?

Given the negative feedback loop in which HES7 protein represses its own transcription, researchers may need to detect both protein presence and active transcription. Recommended approaches include:

• Sequential detection on adjacent sections:

  • Process one section with anti-HES7-HRP antibody for protein detection

  • Process adjacent section with intron probes (e.g., the first intron of HES7) to detect nascent transcripts in the nucleus

• Dual fluorescence approach:

  • Use anti-HES7 antibody with fluorescent secondary antibody for protein detection

  • Combine with RNA FISH using intronic probes labeled with a different fluorophore

  • This allows visualization of regions where protein is present but transcription is repressed

• Interpreting results:

  • Expect HES7 transcription to occur primarily in HES7 protein-negative domains

  • Anticipate constitutive up-regulation of HES7 transcription in the absence of HES7 protein

  • Consider using Hes7 essential region (−1.5 to −1.1 kb) reporters to track transcriptional activity

What are common issues when using HES7 antibody-HRP conjugates and how can they be resolved?

How does the conjugation process affect antibody specificity and sensitivity?

Researchers should consider several factors that influence conjugate performance:

• Epitope masking: The conjugation process attaches HRP to surface-exposed lysine residues. If lysine residues are present in or near the antigen-binding site, conjugation may affect binding activity .

• Degree of labeling:

  • Too few HRP molecules per antibody reduces sensitivity

  • Too many HRP molecules per antibody may interfere with antigen binding

  • Enhanced methods (like lyophilization during conjugation) can increase the number of HRP molecules bound to each antibody without compromising specificity

• Purification considerations:

  • Removing unconjugated HRP improves the immunohistoenzymic properties of conjugates

  • Both Sephadex G-200 gel chromatography and ammonium sulfate precipitation have been used effectively

  • Ensure conjugated and unconjugated IgG are properly separated

• Validation approaches:

  • Test conjugate functionality with known positive samples at various dilutions

  • Compare signal intensity and specificity between direct conjugates and indirect detection methods

  • Evaluate specificity through absorption controls using the immunizing peptide

How can researchers study HES7's role in transcriptional regulation using HRP-conjugated antibodies?

HES7 functions as a transcriptional repressor, making it important to study its regulatory activities:

• Reporter assays:

  • Co-transfect cells with a luciferase vector under the control of the HES7 promoter (0.9-kb) and the HES7 expression vector

  • Measure how HES7 represses its own promoter activity

  • Include constitutively active Notch (caNotch) to study how HES7 overrides Notch-induced transcription

• Regulatory element analysis:

  • The HES7 essential region (−1.5 to −1.1 kb) contains several critical regulatory elements:

    • Three T-boxes (YMACACYY or complementary)

    • Six E-boxes (CANNTG)

    • One RBP-J-binding site (YRTGDGAD or complementary)

  • Mutational analysis has demonstrated that E-boxes are essential for HES7 expression in the PSM, while T-boxes play an auxiliary role

• Transcription factor interactions:

  • HES7 expression is activated by a synergistic effect of multiple factors:

    • Mesogenin1 (binding to E-boxes)

    • Tbx6 (binding to T-boxes)

    • Notch signaling (via the RBP-J binding element)

  • HRP-conjugated antibodies can be used in co-immunoprecipitation experiments to study these interactions

What emerging technologies might enhance the use of HES7 antibody-HRP conjugates in research?

Researchers should consider these innovative approaches:

• High-density hapten labeling:

  • Adding multiple detectable haptens (16-32 per oligonucleotide) can improve detection sensitivity

  • Direct HRP conjugation to oligonucleotide probes has shown even greater sensitivity than multi-hapten versions

  • These approaches can be applied to in situ hybridization for detecting HES7 mRNA in cells and tissues

• Advanced conjugation chemistries:

  • Site-specific conjugation methods that avoid the antigen-binding region

  • Engineered antibody variants with additional conjugation sites away from the binding domain

  • Oriented conjugation techniques to ensure optimal antigen binding

• Multiplexed detection systems:

  • Combining HRP-conjugated HES7 antibodies with antibodies against other factors in the segmentation clock

  • Using different chromogenic substrates for simultaneous visualization of multiple targets

  • Integrating with advanced imaging techniques like lightsheet microscopy for in vivo studies

How might understanding HES7 contribute to broader research in developmental biology and disease models?

The study of HES7 using HRP-conjugated antibodies has implications for several research areas:

• Developmental timing mechanisms:

  • Further understanding of the segmentation clock that controls somite formation

  • Insights into how oscillatory gene expression drives developmental processes

  • Investigation of other negative feedback loops in embryonic patterning

• Disease modeling:

  • HES7 mutations cause spondylocostal dysostosis type 4 (SCDO4)

  • Research on how specific mutations affect protein function and stability

  • Development of therapeutic approaches targeting the Notch-HES7 pathway

• Stem cell differentiation:

  • Application of knowledge about HES7's role in directing PSM-specific expression

  • Engineering of stem cell differentiation protocols for generating somitic tissue

  • Creation of reporter systems based on the HES7 essential region to track differentiation