SHC1 Antibody, Biotin conjugated

What is SHC1 and what are its major isoforms?

SHC1 (SHC-transforming protein 1) is a ubiquitously-expressed signaling adapter protein that links activated growth factor receptors to downstream signaling pathways. It exists in three functionally-distinct isoforms with molecular weights of 46 kDa, 52 kDa, and 66 kDa. These isoforms are encoded from the same gene and share a common structure, with each possessing several phosphorylation sites . The most significant role of SHC1 is in mediating EGFR signaling through phosphorylation events and protein interactions, which define output from the EGFR pathway . In experimental contexts, all three isoforms can be detected via Western blotting, appearing as bands at approximately 51 kDa, 55 kDa, and 66 kDa .

Why use biotin-conjugated antibodies for SHC1 detection?

Biotin-conjugated antibodies offer significant advantages for SHC1 detection in research applications. The biotin-avidin/streptavidin interaction is characterized by exceptional binding strength with a dissociation constant (Kd) of 4 × 10^-14 M , providing stable detection complexes. This strong interaction enables various experimental strategies including signal amplification, which is particularly valuable when detecting proteins like SHC1 that may exist in multiple isoforms or at varying expression levels. Additionally, the biotin-conjugation allows for flexible detection systems as researchers can use different streptavidin-conjugated reporter molecules (HRP, fluorophores, etc.) with the same primary antibody preparation. For multiplex experiments, biotin-conjugated antibodies can be particularly useful when combined with other detection methods .

What are the recommended storage conditions for SHC1 Antibody (Biotin conjugated)?

For optimal performance and longevity, SHC1 Antibody (Biotin conjugated) should be stored at 4°C in the dark . Some products may be stored at -20°C or below, depending on the manufacturer's specifications . The dark storage condition is particularly important for biotin-conjugated antibodies to prevent photobleaching of the biotin moiety. Most preparations contain preservatives such as sodium azide (typically 0.02-0.05%) and may include stabilizers like BSA (0.25%) and glycerol (50%) . Before using the antibody after storage, allow it to equilibrate to room temperature and briefly centrifuge to collect the solution at the bottom of the vial. Avoid repeated freeze-thaw cycles as they can damage the antibody and reduce its effectiveness.

What are the validated applications for SHC1 Antibody (Biotin conjugated) and optimal dilutions?

Based on the current literature and product specifications, SHC1 Antibody (Biotin conjugated) has been validated for several research applications with specific optimal dilution ranges:

The optimal dilution should be experimentally determined for each specific application and experimental system. For ELISA applications, a 100-fold dilution (10 μl of biotin-antibody + 990 μl of biotin-antibody diluent) is typically recommended as a starting point .

How should I design experiments to detect specific SHC1 isoforms?

When designing experiments to detect specific SHC1 isoforms (p46, p52, and p66), several methodological considerations are essential:

• Antibody selection: Verify that your biotin-conjugated SHC1 antibody recognizes the specific isoform of interest. Some antibodies detect all isoforms, while others are isoform-specific. For example, an antibody targeting the immunogen region between residue 300 and 350 will detect different isoforms than one targeting residues 484-583 .

• Resolution optimization: For Western blot applications, use a lower percentage acrylamide gel (8-10%) to achieve better separation of the three isoforms, which have similar molecular weights. Extended run times will further improve separation.

• Control lysates: Include positive control lysates known to express specific isoforms. For example, HeLa cells express all three major isoforms and can serve as a reference standard showing bands at approximately 51 kDa, 55 kDa, and 66 kDa .

• Phosphorylation status: Consider whether phosphorylated forms need to be detected, as phosphorylation can alter migration patterns of SHC1 isoforms. Use phosphatase treatments as controls if necessary.

• Quantification strategy: When quantifying relative isoform expression, normalize each isoform to an appropriate loading control and analyze them separately, rather than combining signal from all isoforms.

What is the detection limit for SHC1 using biotin-conjugated antibodies in ELISA?

The detection limit for SHC1 using biotin-conjugated antibodies in ELISA applications is typically very sensitive. According to standard ELISA kit specifications for SHC1, the detection range is 25-1600 pg/ml with a minimum detectable dose (sensitivity) of less than 6.25 pg/ml . This sensitivity is defined as the lowest protein concentration that can be differentiated from zero, determined by the mean optical density value of zero standard replicates plus three standard deviations.

To achieve this level of sensitivity in experimental settings:

• Use freshly prepared reagents and follow the recommended 100-fold dilution protocol for biotin-conjugated antibodies (10 μl antibody + 990 μl diluent) .

• Ensure proper blocking to minimize background signal.

• Optimize incubation times and washing steps to maximize signal-to-noise ratio.

• Consider using streptavidin-HRP with enhanced chemiluminescent substrates for detection near the lower limit.

• Include a standard curve with at least 7 points within the detection range (25-1600 pg/ml) to accurately quantify SHC1 concentrations in unknown samples.

How can I minimize background staining when using biotin-conjugated SHC1 antibodies?

Background staining is a common challenge when using biotin-conjugated antibodies. To minimize this issue:

• Choose appropriate conjugation methods: The ZBPA conjugation method (utilizing modified Z-domain of protein A) results in more specific targeting of the Fc part of antibodies compared to general chemical conjugation kits like Lightning-Link. Studies show ZBPA biotinylation produces distinct immunoreactivity without off-target staining, whereas other methods may display nonspecific staining patterns .

• Block endogenous biotin: Tissues and cells can contain endogenous biotin that causes background. Pretreat samples with avidin/biotin blocking kits before applying the biotin-conjugated primary antibody.

• Optimize antibody concentration: Titrate the antibody to find the optimal concentration that gives specific signal with minimal background. Excessive antibody concentrations often lead to increased background.

• Extended washing steps: Implement longer and more numerous washing steps with appropriate buffers (PBS-T or TBS-T) to remove unbound antibody.

• Use appropriate diluents: Dilute antibodies in solutions containing 0.25% BSA and 0.02% sodium azide to reduce nonspecific binding .

• Pre-adsorb antibodies: For polyclonal biotin-conjugated SHC1 antibodies, consider pre-adsorbing against tissues or cells lacking SHC1 to remove antibodies that might cause cross-reactivity.

What controls should I include when using SHC1 Antibody (Biotin conjugated)?

Robust experimental design requires appropriate controls when using biotin-conjugated SHC1 antibodies:

These controls should be processed identically to experimental samples and included in every experiment to ensure reliable interpretation of results.

How do I troubleshoot weak or absent signal with biotin-conjugated SHC1 antibodies?

When experiencing weak or absent signals when using biotin-conjugated SHC1 antibodies, systematically address the following methodological aspects:

• Antibody activity: Verify antibody activity; some SHC1 antibodies may show negative results in certain applications like Western Blot despite being validated for other techniques .

• Epitope accessibility: The epitope recognized by the antibody (e.g., region between residues 300-350 in human SHC1) may be masked by fixation, denaturation, or protein interactions. Consider alternative sample preparation methods or different antibodies targeting other regions .

• Protein expression levels: Confirm SHC1 expression in your samples. The minimum detectable concentration in optimized ELISA formats is approximately 6.25 pg/ml , but other techniques may have different detection limits.

• Signal amplification: Implement signal amplification methods such as:

  • Tyramide signal amplification for immunohistochemistry

  • Extended substrate incubation times for HRP-based detection

  • Using more sensitive detection reagents

• Biotin-streptavidin system optimization: Ensure your streptavidin-reporter conjugate is functional and used at optimal concentration. Consider extending the streptavidin-conjugate incubation time.

• Sample preparation: Optimize antigen retrieval methods for fixed tissues or ensure proper protein extraction and denaturation for Western blotting applications.

• Antibody dilution: The recommended dilution may need adjustment; try a series of dilutions to determine the optimal concentration for your specific experimental conditions.

How can I use SHC1 Antibody (Biotin conjugated) to investigate signaling pathway crosstalk?

SHC1 functions as a critical adapter protein in multiple signaling pathways, making it an excellent target for investigating pathway crosstalk. To leverage biotin-conjugated SHC1 antibodies for this purpose:

• Co-immunoprecipitation studies: Use the biotin-conjugated SHC1 antibody to pull down SHC1 and its interacting partners . Analyze the precipitated complex by mass spectrometry or Western blotting for specific pathway components to identify novel interactions.

• Proximity ligation assays: Combine the biotin-conjugated SHC1 antibody with antibodies against proposed interaction partners and use streptavidin-coupled oligonucleotides for proximity ligation assays to visualize protein-protein interactions in situ.

• Phosphorylation dynamics: SHC1 contains multiple phosphorylation sites that are differentially regulated in response to various stimuli. Use the biotin-conjugated SHC1 antibody to immunoprecipitate SHC1 after specific pathway stimulation, then probe with phospho-specific antibodies to map pathway-specific phosphorylation events.

• Temporal analysis: Investigate the dynamics of SHC1 involvement in different pathways by performing time-course experiments following stimulation. This can reveal the sequence of pathway activation and potential feedback mechanisms.

• Subcellular localization studies: Combine the biotin-conjugated SHC1 antibody with streptavidin-fluorophores and markers for different cellular compartments to track SHC1 translocation during signaling events.

This methodological approach can reveal how SHC1 mediates crosstalk between EGFR signaling and other pathways, particularly in cancer contexts where SHC1 plays an oncogenic role .

What are the methodological differences between detecting different SHC1 isoforms in cancer research?

The three SHC1 isoforms (p46, p52, and p66) have distinct roles in cancer biology, necessitating specific methodological approaches for their investigation:

When investigating these isoforms in cancer research:

• Isoform specificity: Ensure the biotin-conjugated SHC1 antibody recognizes the specific isoform of interest. Some antibodies detect all isoforms while others are isoform-specific. Check if the immunogen maps to a region found in all isoforms (e.g., residues 300-350) or is isoform-specific .

• Expression analysis: Different cancer types and stages may express varying levels of each isoform. Use quantitative Western blotting with careful molecular weight discrimination to profile isoform expression patterns across cancer samples.

• Functional studies: When studying isoform-specific functions:

  • Use isoform-specific siRNAs or CRISPR targeting

  • Perform rescue experiments with individual isoforms

  • Consider post-translational modifications unique to each isoform

• Tissue-specific considerations: The p52 isoform's role in breast cancer initiation suggests particular attention to this isoform in breast tissue studies, while other cancer types may show different isoform dynamics.

• Interaction partners: Each isoform may interact with different signaling partners. Use the biotin-conjugated antibody for co-immunoprecipitation followed by mass spectrometry to identify isoform-specific interactomes.

How can I optimize multiplex assays incorporating biotin-conjugated SHC1 antibodies?

Multiplex assays that incorporate biotin-conjugated SHC1 antibodies with other detection systems require careful optimization to prevent cross-reactivity and signal interference:

• Antibody conjugation quality: Use specific biotinylation methods like the ZBPA conjugation approach that targets the Fc portion of antibodies, which has been shown to produce more specific staining compared to general chemical conjugation methods .

• Sequential detection strategy:

  • Apply biotin-conjugated SHC1 antibody first

  • Complete the streptavidin-based detection

  • Block any remaining biotin/streptavidin binding sites

  • Proceed with subsequent antibody staining using non-biotin detection systems

• Spectral separation: When using fluorescent detection systems:

  • Select streptavidin conjugates with fluorophores spectrally distant from other planned fluorophores

  • Perform single-color controls to establish spectral unmixing parameters

  • Consider linear unmixing algorithms for closely spaced emission spectra

• Enzymatic detection optimization: If using HRP-based detection:

  • Use different chromogens for biotin-streptavidin versus other detection systems

  • Implement sequential substrate development with quenching steps between detections

  • Consider tyramide signal amplification with different fluorophores for multiplexing

• Validation controls: Include comprehensive controls for each detection system separately and in combination to identify any cross-reactivity or interference.

• Quantification strategy: Develop a quantification approach that accounts for potential variations in detection efficiency between different multiplexed targets.

Successful multiplex assays can reveal relationships between SHC1 and other proteins in the same cellular contexts, providing valuable insights into complex signaling networks in both normal and disease states.

What are the challenges in interpreting SHC1 phosphorylation data using biotin-conjugated antibodies?

Interpreting SHC1 phosphorylation data presents several methodological challenges when using biotin-conjugated antibodies:

• Multiple phosphorylation sites: SHC1 contains multiple phosphorylation sites that can be differentially regulated. When using general SHC1 antibodies (biotin-conjugated), additional phospho-specific antibodies are needed to determine which sites are modified.

• Isoform-specific phosphorylation: The three SHC1 isoforms may be phosphorylated differently under the same conditions. Since biotin-conjugated SHC1 antibodies often recognize all isoforms , careful molecular weight discrimination is necessary to determine isoform-specific phosphorylation patterns.

• Phosphorylation dynamics: SHC1 phosphorylation is often transient and context-dependent. Consider these methodological approaches:

  • Implement precise time-course experiments with rapid sample fixation

  • Use phosphatase inhibitors during sample preparation

  • Compare results across multiple experimental models to establish consistent patterns

• Quantification challenges: When quantifying phosphorylation levels:

  • Express results as the ratio of phosphorylated to total SHC1 protein

  • Account for baseline phosphorylation in control conditions

  • Consider that some phosphorylation events may alter antibody binding affinity

• Functional correlation: Establishing the functional significance of observed phosphorylation requires additional experiments:

  • Combine with site-directed mutagenesis of phosphorylation sites

  • Correlate phosphorylation with downstream pathway activation

  • Use phosphomimetic mutations to confirm functional outcomes

• Technical artifacts: Phosphorylation detection can be affected by:

  • Antibody binding may be sterically hindered by neighboring phosphorylation events

  • Certain fixation methods may preserve some phosphorylation sites better than others

  • The biotin moiety might affect antibody access to closely positioned phosphorylation sites

Careful experimental design with appropriate controls and validation using complementary methods can help overcome these challenges and yield meaningful insights into SHC1 phosphorylation biology.

How can SHC1 Antibody (Biotin conjugated) be used in studying the role of SHC1 in cancer progression?

SHC1 plays an oncogenic role in several cancer types , making it an important target for cancer research. Biotin-conjugated SHC1 antibodies can be employed in several methodological approaches to study its role in cancer progression:

• Tissue microarray analysis: Apply biotin-conjugated SHC1 antibodies to tissue microarrays containing samples from different cancer stages to correlate SHC1 expression patterns with disease progression. The biotin-streptavidin detection system offers advantages for this high-throughput application due to its sensitivity and low background when properly optimized .

• Isoform-specific investigation: Focus on the p52 isoform which has been specifically implicated in breast cancer initiation . Use biotin-conjugated antibodies that can discriminate between isoforms or combine with precise molecular weight separation techniques.

• Signaling pathway analysis: Implement co-immunoprecipitation with biotin-conjugated SHC1 antibodies followed by mass spectrometry to map the SHC1 interactome in normal versus cancer cells, revealing potential cancer-specific interactions.

• Patient-derived xenograft models: Apply biotin-conjugated SHC1 antibodies in immunohistochemical analysis of patient-derived xenograft models to:

  • Track changes in SHC1 expression during tumor evolution

  • Correlate SHC1 expression with treatment response

  • Identify potential biomarkers for patient stratification

• Circulating tumor cell analysis: Develop protocols using biotin-conjugated SHC1 antibodies for the detection of SHC1 in circulating tumor cells, potentially offering a liquid biopsy approach for monitoring disease.

• Combination with genomic data: Correlate protein-level findings using biotin-conjugated SHC1 antibodies with genomic alterations to build integrated models of SHC1's role in cancer progression.

This multi-faceted approach can provide insights into how SHC1 contributes to cancer progression and potentially identify novel therapeutic targets within SHC1-mediated signaling pathways.

What methodological approaches can address the discrepancies in SHC1 antibody results across different studies?

Research involving SHC1 antibodies sometimes yields discrepancies across studies. To address these methodological challenges:

• Standardized reporting: Implement comprehensive antibody reporting including:

  • Specific clone or catalog number

  • Immunogen information (e.g., "immunogen mapping to region between residue 300 and 350" )

  • Biotinylation method used (ZBPA vs. chemical conjugation)

  • Lot number and validation data

  • Detailed experimental protocols

• Cross-validation strategies:

  • Use multiple antibodies targeting different epitopes of SHC1

  • Confirm key findings with genetic approaches (siRNA, CRISPR)

  • Implement orthogonal detection methods (mass spectrometry)

  • Share positive control lysates between laboratories

• Technical standardization:

  • Establish reference standards for the three SHC1 isoforms

  • Develop consensus protocols for sample preparation

  • Create detailed guidelines for image acquisition and quantification

  • Use digital pathology tools for more objective scoring

• Antibody validation rigor:

  • Perform knockout/knockdown validation for each new lot

  • Test for cross-reactivity with closely related proteins

  • Verify epitope specificity with peptide competition assays

  • Assess performance across multiple applications and fixation methods

• Reproducibility initiatives:

  • Establish multi-laboratory validation studies

  • Create repositories of validated protocols

  • Develop automated analysis pipelines to reduce subjective interpretation

By implementing these methodological approaches, researchers can reduce discrepancies and build a more coherent understanding of SHC1 biology across different experimental systems and disease contexts.

How can novel biotinylation methods improve SHC1 detection specificity and sensitivity?

Recent advances in antibody biotinylation methods offer opportunities to enhance both the specificity and sensitivity of SHC1 detection:

• Site-specific conjugation: The ZBPA conjugation method, which utilizes a modified Z-domain of protein A to specifically target the Fc part of antibodies, has demonstrated superior performance compared to general chemical conjugation kits like Lightning-Link . This method produces distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the antibody buffer.

• Optimized biotin-to-antibody ratio: Controlling the biotin-to-antibody ratio is critical for:

  • Preventing over-biotinylation that can compromise antigen binding

  • Ensuring sufficient biotin molecules for detection

  • Maintaining proper antibody folding and function

• Enzyme-mediated biotinylation: Enzymatic approaches using biotin ligase (BirA) can achieve site-specific biotinylation at engineered recognition sequences, offering superior control compared to chemical methods.

• Click chemistry approaches: Two-step labeling methods using click chemistry can separate the antibody modification step from the biotin attachment step, reducing interference with antigen binding:

  • First introduce azide or alkyne groups to the antibody

  • Then attach biotin through highly specific click reactions

• Nanobody and recombinant antibody fragments: Consider using biotinylated nanobodies or recombinant antibody fragments specific to SHC1 for:

  • Better tissue penetration

  • Reduced background from Fc interactions

  • More consistent biotinylation stoichiometry

• Verification methodologies: To ensure specificity of newly biotinylated antibodies:

  • Compare staining patterns with non-biotinylated versions

  • Perform parallel validations on cells with manipulated SHC1 expression

  • Analyze multiple lots for consistency

Implementation of these advanced biotinylation approaches can significantly improve SHC1 detection, particularly in challenging applications like detecting specific isoforms or phosphorylation states in complex tissue environments.

What are the key considerations for selecting the optimal biotin-conjugated SHC1 antibody for specific research applications?

When selecting a biotin-conjugated SHC1 antibody for specific research applications, researchers should consider several critical factors:

• Epitope specificity: Verify which region of SHC1 the antibody recognizes (e.g., region between residues 300-350 vs. residues 484-583 ) and how this relates to your research question. Different epitopes may be differentially accessible depending on experimental conditions.

• Isoform detection: Determine whether the antibody detects all three SHC1 isoforms (p46, p52, p66) or is specific to particular isoforms. This is especially important when studying isoform-specific functions, such as p52's role in breast cancer initiation .

• Biotinylation method: Consider antibodies biotinylated using the ZBPA method which targets the Fc portion, as these have demonstrated superior specificity compared to general chemical conjugation methods .

• Validated applications: Select antibodies specifically validated for your application of interest. Some SHC1 antibodies work well for immunoprecipitation but show negative results in Western blot .

• Species cross-reactivity: Confirm reactivity with your species of interest. Some SHC1 antibodies are validated for human samples but only predicted to work in mouse, rat, or primate models .

• Quality control data: Review the manufacturer's validation data, including:

  • Specificity testing

  • Lot-to-lot consistency evaluations

  • Application-specific performance metrics

  • Recommended working concentrations

By carefully evaluating these factors, researchers can select the most appropriate biotin-conjugated SHC1 antibody for their specific experimental needs, enhancing the reliability and reproducibility of their results.

How might SHC1 antibody technologies evolve in the next decade of biomedical research?

The evolution of SHC1 antibody technologies over the next decade is likely to incorporate several emerging trends in antibody development and application:

• Single-cell analysis capabilities: Development of highly sensitive biotin-conjugated SHC1 antibodies compatible with single-cell proteomics and single-cell spatial transcriptomics will enable unprecedented resolution of SHC1 function in heterogeneous tissues.

• Engineered recombinant formats: Transition from polyclonal antibodies to fully recombinant SHC1 antibodies with:

  • Defined sequences for improved reproducibility

  • Engineered affinity and specificity

  • Site-specific biotinylation for consistent performance

  • Humanized versions for potential therapeutic applications

• Multimodal detection systems: Development of SHC1 antibodies with dual or multiple labeling beyond biotin:

  • Photo-activatable groups for super-resolution microscopy

  • Mass cytometry tags for high-dimensional analysis

  • DNA barcodes for sequencing-based detection and spatial mapping

• Isoform-specific tools: Creation of highly specific reagents for each SHC1 isoform to better understand their distinct roles in normal physiology and disease states, particularly in cancer where isoform-specific functions have been identified .

• AI-assisted antibody design: Implementation of computational approaches to:

  • Predict optimal epitopes for SHC1 detection

  • Design antibodies with minimal cross-reactivity

  • Optimize biotinylation sites for maximal performance

  • Reduce batch-to-batch variation

• In vivo imaging applications: Development of biotin-conjugated SHC1 antibodies or fragments suitable for in vivo imaging applications with appropriate delivery systems and compatible imaging modalities.

• Therapeutic potential: Exploration of SHC1-targeting antibodies not just as research tools but as potential therapeutics, particularly given SHC1's established oncogenic role in several cancer types .