HES7 Antibody, Biotin conjugated

What is HES7 and why is it significant for developmental biology research?

HES7 (Hairy and Enhancer of Split 7) is a transcription factor belonging to the basic helix-loop-helix (bHLH) family of proteins. It is primarily expressed in the presomitic mesoderm (PSM) and functions as a transcriptional repressor that binds to N-box or E-box elements in promoters of target genes. HES7 plays a crucial role in vertebrate somitogenesis through its cyclical expression pattern, which occurs in approximately two-hour cycles. This oscillatory expression is essential for proper segmentation of somites from the presomitic mesoderm . Researchers studying developmental processes, particularly those related to spine, rib, and vertebral formation, often investigate HES7 due to its association with disorders like spondylocostal dysostosis when mutated .

How does HES7 function in the molecular clock mechanism during somitogenesis?

HES7 functions as a key component of the segmentation clock through an auto-regulatory negative feedback loop. The HES7 protein represses its own transcription by binding to its promoter region, as confirmed by chromatin immunoprecipitation (ChIP) studies . This creates an oscillatory pattern where:

• HES7 mRNA is transcribed

• HES7 protein accumulates

• HES7 protein inhibits its own transcription

• HES7 protein undergoes proteasome-mediated degradation

• Inhibition is relieved, allowing the cycle to restart

This oscillatory expression is critical for somite segmentation. Additionally, HES7 represses Lunatic Fringe (LFNG) expression, thereby regulating the Notch signaling pathway both directly and indirectly . When HES7 function is disrupted, somitogenesis is abnormal, leading to vertebral malformations.

Why would researchers use biotin-conjugated antibodies for HES7 studies?

Biotin conjugation offers several methodological advantages for researchers studying cyclically expressed proteins like HES7:

• Signal amplification: The biotin-streptavidin system provides significant signal enhancement due to the multiple biotin molecules that can be conjugated to a single antibody and the high binding affinity between biotin and streptavidin

• Detection sensitivity: For proteins with fluctuating expression levels like HES7, the amplification helps detect lower abundance protein during certain phases of the expression cycle

• Versatility: Biotin-conjugated antibodies can be used with various detection systems including fluorescent, chromogenic, or chemiluminescent streptavidin conjugates

• Multiplexing capability: When studying multiple components of the Notch pathway simultaneously with HES7

The streptavidin-biotin binding is extremely strong (almost as strong as a covalent bond) due to the large number of hydrogen bonds formed between them, making it one of the strongest non-covalent interactions in biological systems .

What are the typical applications for HES7 antibodies in developmental research?

HES7 antibodies are used in several applications within developmental biology research:

• Immunohistochemistry/Immunofluorescence: To visualize HES7 protein localization in the presomitic mesoderm and track its dynamic expression patterns

• Western blotting: To quantify HES7 protein levels and confirm specificity

• Chromatin Immunoprecipitation (ChIP): To identify HES7 binding sites on target promoters, including its own promoter and the LFNG promoter

• Developmental timing studies: To track the oscillatory expression patterns of HES7 during somite formation

• Mutant phenotype analysis: To analyze alterations in HES7 expression and localization in disease models

What sample types can be effectively analyzed using HES7 antibodies?

Based on available research data, HES7 antibodies have been successfully used with:

• Mouse embryonic tissue sections, particularly presomitic mesoderm (PSM)

• Whole-mount immunostaining of vertebrate embryos

• Cell culture systems expressing wildtype or mutant HES7

• Chromatin preparations from PSM tissues for ChIP analysis

What are optimal protocols for using biotin-conjugated HES7 antibodies in immunohistochemistry?

When conducting immunohistochemistry with biotin-conjugated HES7 antibodies, researchers should consider the following protocol guidelines:

• Tissue preparation: Fix tissues in 4% paraformaldehyde for 2-4 hours (embryonic tissues) or overnight (adult tissues)

• Antigen retrieval: Perform heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Critical pre-blocking step: Block endogenous biotin before applying primary antibodies to prevent non-specific binding using a biotin blocking protocol

• Antibody dilution: Typically use at 1:20-1:50 dilution range for immunohistochemistry (adjust based on specific antibody specifications)

• Detection: Apply streptavidin-conjugated reporter molecules (fluorescent, enzymatic, or metal-based)

• Controls: Include tissues from HES7-knockout models as negative controls

How should biotin blocking be performed when using biotin-conjugated HES7 antibodies?

Biotin blocking is a critical step when using biotin-conjugated antibodies to reduce background signal. The protocol should be performed after normal serum blocking but before primary antibody incubation:

• Wash tissue sections with appropriate buffer (PBS or TBS)

• Prepare fresh 0.01% biotin solution in wash buffer

• Apply biotin solution to tissues and incubate for 15 minutes at room temperature

• Wash thoroughly with buffer

• Apply streptavidin solution and incubate for 15 minutes

• Wash thoroughly before proceeding to primary antibody incubation

Commercial biotin blocking kits are also available as alternatives to this protocol.

How can I detect oscillatory expression patterns of HES7 in presomitic mesoderm?

Detecting the oscillatory expression of HES7 requires careful timing and preparation:

• Sample collection: Collect embryos at closely spaced time intervals to capture different phases of the 2-hour oscillation cycle

• Whole-mount approach: Use whole-mount immunostaining to visualize the dynamic patterns across the entire PSM

• Pattern identification: Compare specimens to identify the three main expression patterns:

  • Pattern 1: HES7 immunoreactivity mainly in the middle PSM

  • Pattern 2: Strong HES7 immunoreactivity in both posterior and anterior PSM

  • Pattern 3: Signal extending from posterior to middle PSM with weakening anterior signal

• Co-detection: Consider co-staining for LFNG to observe the complementary expression patterns, as HES7 and LFNG transcription occurs in HES7 protein-negative domains

• Time-lapse imaging: For live imaging studies, use reporter constructs with the 0.9-kb HES7 promoter which contains regions necessary for cyclic expression

What are appropriate controls when using biotin-conjugated HES7 antibodies?

Proper controls are essential for validating HES7 antibody specificity and signal accuracy:

What detection systems work best with biotin-conjugated HES7 antibodies?

Several detection systems can be used effectively with biotin-conjugated antibodies:

• Streptavidin-HRP followed by DAB: Provides brown precipitate suitable for bright-field microscopy and long-term storage

• Streptavidin-conjugated fluorophores: Offers higher sensitivity and multiplexing capabilities

• Streptavidin-alkaline phosphatase: Alternative chromogenic detection with different color options

• Tyramide signal amplification (TSA): Provides further amplification for very low abundance targets

For the specific detection of oscillatory HES7 expression patterns, fluorescent detection systems often provide superior results due to their higher sensitivity and dynamic range.

How can I analyze HES7 binding to its own promoter and other target genes?

Chromatin immunoprecipitation (ChIP) is the primary method for analyzing HES7 binding to target promoters:

• Sample preparation: Isolate and cross-link PSM tissues from appropriate developmental stages

• Chromatin fragmentation: Sonicate to achieve fragments of approximately 200-500 bp

• Immunoprecipitation: Use specific anti-HES7 antibodies (can be biotin-conjugated)

• Analysis: Perform qPCR targeting regions of interest:

  • The HES7 promoter region, which contains N-box or E-box elements

  • The LFNG promoter region, which is directly regulated by HES7

• Controls: Include input chromatin samples and immunoprecipitation with preimmune serum

Research has confirmed that anti-HES7 antibodies specifically precipitate chromatin containing both HES7 and LFNG promoter regions from PSM tissues, demonstrating direct interaction between HES7 protein and these regulatory regions in vivo .

How can experimental design address the transient nature of HES7 expression?

The oscillatory expression of HES7 presents unique experimental challenges:

• Timed sample collection: Harvest samples at precise intervals (15-20 minute increments) to capture different phases of the oscillation cycle

• Protein stabilization approaches: Use proteasome inhibitors to transiently stabilize HES7 protein for certain applications

• Reporter systems: Utilize the HES7 promoter (0.9-kb) driving fluorescent protein expression to monitor dynamics in real-time

• Computational modeling: Integrate experimental data with mathematical models of the oscillatory network

• Genetic manipulations: Study HES7BAP/BAP knock-in mice that show impaired oscillatory behavior as research tools

Research with the HES7BAP variant (containing a 14-amino acid biotin acceptor peptide) shows that even though this modified protein retains wildtype-like repressor activity in cultured cells, it displays greatly impaired activity in vivo, leading to non-oscillatory expression. This highlights the sensitivity of the oscillatory mechanism to subtle protein modifications .

What experimental approaches can distinguish between HES7 and other HES family members?

Distinguishing between highly similar HES family proteins requires careful experimental design:

• Antibody selection: Use antibodies raised against unique regions of HES7, particularly those targeting the PPPPHSQDGAPKAPLPPPPAFWRPWP sequence

• Expression pattern analysis: HES7 has a distinct expression pattern confined to the PSM, while other HES family members may have broader expression domains

• Temporal dynamics: Focus on the oscillatory nature of HES7 expression, which differs from some other family members

• Loss-of-function controls: Include tissue samples from HES7-null mice to confirm antibody specificity

• Peptide competition assays: Perform parallel staining with antibody pre-absorbed with immunizing peptide

How can I study the relationship between HES7 and Notch signaling components?

Investigating the interplay between HES7 and the Notch pathway requires specialized approaches:

• Co-immunoprecipitation: Determine whether HES7 physically interacts with Notch pathway components

• Dual reporter systems: Use fluorescent reporters for both HES7 and Notch activity to track temporal relationships

• Genetic interaction studies: Analyze compound mutants affecting both HES7 and Notch pathway components

• Sequential ChIP: Determine whether HES7 and Notch intracellular domain (NICD) co-occupy certain genomic regions

• Target gene analysis: Measure expression of genes regulated by both pathways, particularly LFNG, which shows non-oscillatory expression throughout the PSM in HES7BAP/BAP embryos

Why might I observe high background when using biotin-conjugated HES7 antibodies?

High background is a common challenge with biotin-conjugated antibodies due to several factors:

• Endogenous biotin: Tissues naturally contain biotin, which can bind streptavidin detection reagents

• Incomplete biotin blocking: Insufficient blocking of endogenous biotin

• Non-specific binding: Primary antibody binding to non-target proteins

• Excessive antibody concentration: Using too concentrated antibody solutions

• Suboptimal washing: Inadequate washing between steps

Recommendations to overcome these issues include:

• Implement thorough biotin blocking protocols using biotin followed by streptavidin before primary antibody application

• Optimize antibody dilutions (typically starting with 1:20-1:50 for immunohistochemistry)

• Include additional blocking proteins (such as BSA or serum)

• Extend washing steps with agitation

• Consider alternative detection systems if background persists

What strategies can address the challenges of detecting oscillatory protein expression?

Detecting proteins with dynamic expression patterns like HES7 presents unique challenges:

• Synchronization issue: Individual embryos may be at different phases of the oscillation cycle

• Quantification challenges: Need to categorize samples into different expression phases

• Rapid degradation: HES7 protein undergoes proteasome-mediated degradation

Effective strategies include:

• Collect and analyze large sample numbers to capture all oscillation phases

• Categorize samples into the three main expression patterns observed in the PSM

• Use parallel samples for RNA and protein detection to correlate transcription with protein expression

• Consider live imaging approaches with reporter constructs to follow dynamics in real time

• Include additional markers of the segmentation clock to provide context for the expression phase

How do fixation and processing conditions affect HES7 antibody staining?

Fixation and processing conditions significantly impact HES7 detection:

• Fixation timing: Overfixation can mask epitopes, while underfixation can lead to poor tissue preservation

• Fixative selection: Paraformaldehyde (4%) is typically optimal for HES7 detection; avoid Bouin's solution

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer improves detection

• Processing temperature: Maintain consistent temperature during processing to preserve epitope integrity

• Storage effects: Prolonged storage of cut sections can reduce immunoreactivity

For embryonic tissues where HES7 is typically studied, shorter fixation times (2-4 hours) generally yield better results than overnight fixation.

What are the considerations when using HES7 antibodies in disease models?

When studying HES7 in disease contexts, particularly spondylocostal dysostosis:

• Mutation effects: Different mutations may affect antibody epitope recognition

• Expression levels: Disease states may have significantly reduced expression levels requiring more sensitive detection

• Spatial alterations: Altered distribution patterns may require whole-mount approaches

• Protein stability: Some mutations may affect protein stability and degradation rates

• Control selection: Use appropriate controls including wildtype littermates and known pathogenic variants

Research shows that even subtle modifications to HES7, such as the BAP tag insertion, can dramatically affect protein function while retaining immunoreactivity , highlighting the importance of careful interpretation of antibody staining in disease models.

How can I correlate HES7 protein expression with transcriptional activity?

To establish relationships between HES7 protein presence and its transcriptional effects:

• Sequential section analysis: Perform RNA in situ hybridization and protein immunodetection on sequential sections

• Dual labeling approaches: Combine RNA FISH with immunofluorescence when possible

• Reporter assays: Use reporter constructs containing HES7-responsive elements

• ChIP-seq correlation: Compare HES7 binding locations with transcriptional changes

• Temporal analysis: Track the relationship between protein expression and target gene transcription over time