SHROOM2 Antibody, Biotin conjugated

What is the primary structure and function of SHROOM2 protein?

SHROOM2 (Shroom Family Member 2) is a protein-coding gene that functions as a key regulator of cell shape and plays a crucial role in various cellular processes, including cell migration and tissue morphogenesis . It represents the human homolog of Xenopus laevis apical protein (APX) gene implicated in amiloride-sensitive sodium channel activity . The protein contains multiple functional domains, with an observed molecular weight of approximately 180 kDa . SHROOM2 is expressed in endothelial cells and facilitates the formation of a contractile network within these cells . Functionally, it may be involved in endothelial cell morphology changes during cell spreading and, in the retinal pigment epithelium, may regulate the biogenesis of melanosomes and promote their association with the apical cell surface by inducing gamma-tubulin redistribution .

What are the recommended applications for SHROOM2 antibody, biotin conjugated?

The biotin-conjugated SHROOM2 antibody is primarily validated for several experimental applications, including Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry on paraffin-embedded tissues (IHC-P), and Immunohistochemistry on frozen tissues (IHC-fro) . The biotin conjugation facilitates detection using streptavidin-based secondary detection systems, which can provide signal amplification for enhanced sensitivity in these applications. When designing experiments, researchers should implement proper controls and consider the recommended dilution ranges to optimize signal-to-noise ratios for specific applications .

What experimental conditions are optimal for using biotin-conjugated SHROOM2 antibodies in immunohistochemistry?

For optimal immunohistochemistry results using biotin-conjugated SHROOM2 antibodies, a dilution range of 1:200-1:500 is recommended based on validated protocols . The process typically involves antigen retrieval (method depends on tissue fixation), blocking with appropriate serum (typically 10% normal goat serum), and incubation with the primary antibody . Detection should employ a streptavidin-based detection system compatible with biotin conjugation. For paraffin-embedded tissues (IHC-P), complete deparaffinization and rehydration are essential before antigen retrieval . For frozen sections (IHC-fro), fixation conditions should be optimized based on tissue type. Signal development systems should be selected based on the desired detection sensitivity and imaging modality .

What are the binding specificity characteristics of the biotin-conjugated SHROOM2 antibody?

The biotin-conjugated SHROOM2 antibody (AA 213-405) has defined binding specificity targeted to amino acids 213-405 of the SHROOM2 protein . This polyclonal antibody is raised in rabbits against recombinant Human Protein Shroom2 protein (213-405AA) . It demonstrates specific reactivity to human SHROOM2 protein and has been validated through multiple applications, including ELISA and immunohistochemistry . The antibody undergoes protein G purification with >95% purity, which helps ensure specific binding to the target epitope . When conducting experiments, researchers should be aware of the specific binding region to interpret results accurately, particularly when studying protein isoforms or domains.

How does biotin conjugation affect the detection sensitivity and specificity of SHROOM2 antibodies compared to other conjugates?

Biotin conjugation provides several advantages for SHROOM2 antibody detection compared to other conjugates, particularly in terms of signal amplification potential. The high affinity interaction between biotin and streptavidin (Kd ≈ 10^-15 M) enables sensitive detection systems with multiple layers of amplification . In comparison to direct fluorophore conjugates like FITC, biotin-conjugated antibodies often provide greater sensitivity through the streptavidin-biotin amplification system, which is especially valuable when detecting low-abundance proteins like SHROOM2 in certain tissues .

What are the molecular interactions between SHROOM2 and cytoskeletal components that can be investigated using biotin-conjugated antibodies?

Biotin-conjugated SHROOM2 antibodies can be instrumental in investigating the complex molecular interactions between SHROOM2 and cytoskeletal components. Research has demonstrated that SHROOM2 directly interacts with F-actin and can protect actin filaments from cytochalasi . Additionally, SHROOM2 interacts with the C-terminal MyTH4-FERM domain of myosin VIIa .

When designing experiments to study these interactions, co-immunoprecipitation coupled with Western blotting using biotin-conjugated SHROOM2 antibodies can help identify protein-protein interactions in cellular lysates. For visualization of co-localization, immunofluorescence microscopy using biotin-conjugated SHROOM2 antibodies with streptavidin-fluorophore detection, combined with actin filament staining (phalloidin) or myosin VIIa detection, can reveal spatial relationships between these proteins . The biotin-conjugated format is particularly valuable in multi-color immunofluorescence studies as it allows flexible secondary detection strategies.

How can the biotin-conjugated SHROOM2 antibody be utilized to investigate the protein's role in tight junction formation and epithelial morphogenesis?

SHROOM2 has been identified as a submembranous PDZ domain-containing protein associated with tight junctions in multiple embryonic and adult epithelia . To investigate this function, biotin-conjugated SHROOM2 antibodies can be employed in several sophisticated experimental approaches:

• Immunohistochemistry on tissue sections: Using the biotin-conjugated antibody at 1:200-1:500 dilution on tissue sections to visualize SHROOM2 localization at tight junctions across different epithelial tissues .

• Co-localization studies: Combining biotin-conjugated SHROOM2 antibody detection with markers for tight junction proteins (e.g., ZO-1, claudins, occludin) to assess spatial relationships.

• Functional knockdown studies: Utilizing immunofluorescence with biotin-conjugated SHROOM2 antibodies to evaluate tight junction integrity following SHROOM2 knockdown or overexpression in epithelial cell models.

• Live cell imaging: Employing the antibody in permeabilized cells to monitor dynamic changes in SHROOM2 localization during junction formation or epithelial morphogenesis.

The biotin-conjugation offers flexibility in detection strategies, allowing for signal amplification in tissues where SHROOM2 expression may be limited .

What controls should be included when using biotin-conjugated SHROOM2 antibodies in immunoassays?

When designing experiments with biotin-conjugated SHROOM2 antibodies, the following controls are essential for result validation:

These controls are particularly important when using biotin-conjugated antibodies due to potential endogenous biotin interference in certain tissues and the complexity of the multi-step detection process .

What protocols are recommended for using biotin-conjugated SHROOM2 antibody in dual or multi-labeling experiments?

For multi-labeling experiments using biotin-conjugated SHROOM2 antibody alongside other primary antibodies, the following protocol framework is recommended:

• Sample preparation: Fix cells/tissues appropriately (4% paraformaldehyde for cells, tissue-specific methods for sections) .

• Permeabilization: Use 0.2% Triton X-100 for cell permeabilization .

• Blocking: Block with 10% normal goat serum to reduce non-specific binding .

• Endogenous biotin blocking: Apply avidin-biotin blocking kit if using biotin-rich tissues.

• Primary antibody incubation:

  • Sequential approach: Apply biotin-conjugated SHROOM2 antibody (1:200-1:500) first, followed by streptavidin detection, then other primary antibodies.

  • Simultaneous approach: If primary antibodies are from different host species, co-incubate at appropriate dilutions.

• Detection strategy: Use fluorophore-conjugated streptavidin for biotin-SHROOM2 detection and species-specific secondary antibodies for other primaries.

• Counterstaining: Include nuclear counterstain (e.g., DAPI) for context .

This approach minimizes cross-reactivity issues while maximizing detection specificity when examining SHROOM2 alongside other proteins of interest .

How can researchers optimize signal amplification techniques when using biotin-conjugated SHROOM2 antibodies for low-abundance targets?

When detecting low-abundance SHROOM2 in tissues or cells, several signal amplification strategies can be employed with biotin-conjugated antibodies:

• Tyramide Signal Amplification (TSA): This technique can provide 10-100 fold signal enhancement by using streptavidin-HRP followed by biotin-tyramide deposition. The protocol involves:

  • Standard immunostaining with biotin-conjugated SHROOM2 antibody (1:500)

  • Incubation with streptavidin-HRP (1:1000)

  • Brief exposure to biotin-tyramide and H₂O₂

  • Detection with fluorophore-conjugated streptavidin

• Avidin-Biotin Complex (ABC) method: This traditional approach builds multiple layers of signal:

  • Apply biotin-conjugated SHROOM2 antibody

  • Incubate with pre-formed avidin-biotin-HRP complexes

  • Develop with substrate appropriate for microscopy method

• Polymer-based detection systems: These can be combined with biotin-streptavidin for enhanced sensitivity:

  • Use biotin-conjugated SHROOM2 antibody

  • Apply streptavidin-polymer-HRP conjugate

  • Develop with chromogenic or fluorescent substrates

Each method offers different sensitivity levels and should be optimized for specific experimental conditions, with careful attention to background signal .

How can researchers address specificity concerns when interpreting results from biotin-conjugated SHROOM2 antibody experiments?

When evaluating data from experiments using biotin-conjugated SHROOM2 antibodies, researchers should implement the following approaches to verify specificity:

• Correlation with molecular weight: Confirm that detected bands in Western blot match the expected molecular weight of SHROOM2 (approximately 180 kDa observed, though calculated weight is 16799 MW) .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (213-405AA region of SHROOM2) to confirm signal suppression .

• Multiple antibody validation: Compare results with different SHROOM2 antibodies targeting distinct epitopes (e.g., AA 599-625, AA 701-850, or AA 1295-1344) .

• Genetic validation: Use SHROOM2 knockdown or knockout models to verify signal reduction/elimination.

• Subcellular localization assessment: Confirm that staining patterns align with known SHROOM2 localization (e.g., tight junctions in epithelia, association with actin cytoskeleton) .

• Cross-reactivity evaluation: Test the antibody on samples from species not recognized by the antibody to confirm absence of non-specific signals .

• Orthogonal technique validation: Correlate protein detection with mRNA expression data for SHROOM2.

What are the potential artifacts and issues specific to using biotin-conjugated antibodies for SHROOM2 detection, and how can they be mitigated?

Several artifacts can complicate data interpretation when using biotin-conjugated SHROOM2 antibodies:

Proper sample preparation, including appropriate fixation (4% paraformaldehyde), permeabilization (0.2% Triton X-100), and blocking procedures are critical for minimizing these artifacts .

How can biotin-conjugated SHROOM2 antibodies be employed to investigate disease mechanisms associated with SHROOM2 dysfunction?

SHROOM2 has been associated with several diseases, including Meniere Disease and Flinders Island Spotted Fever . Biotin-conjugated SHROOM2 antibodies offer valuable tools for investigating these disease mechanisms through several sophisticated approaches:

• Comparative immunohistochemistry: Using biotin-conjugated SHROOM2 antibodies to compare protein expression and localization patterns between normal and disease tissues at 1:200-1:500 dilution . This approach can reveal alterations in SHROOM2 distribution at tight junctions or associations with the cytoskeleton.

• Protein interaction network analysis: Employing biotin-conjugated antibodies in proximity ligation assays to detect disruptions in SHROOM2 interactions with myosin VIIa or F-actin in disease models .

• Quantitative tissue analysis: Implementing automated image analysis of biotin-streptavidin immunostained tissues to quantify SHROOM2 expression level changes across disease progression stages.

• Functional consequences analysis: Combining SHROOM2 immunodetection with markers of endothelial cell function, as SHROOM2 depletion is known to increase endothelial sprouting, migration, and angiogenesis .

• Therapeutic response monitoring: Using the antibody to assess restoration of normal SHROOM2 distribution following experimental interventions.

These approaches leverage the sensitivity and specificity of biotin-conjugated SHROOM2 antibodies to provide insights into molecular mechanisms underlying disease pathogenesis .

What emerging technologies can be combined with biotin-conjugated SHROOM2 antibodies to advance understanding of SHROOM2 biology?

Several cutting-edge technologies can be integrated with biotin-conjugated SHROOM2 antibodies to drive new discoveries:

• Super-resolution microscopy: Techniques like STORM, PALM, or STED microscopy combined with biotin-conjugated SHROOM2 antibodies can reveal nanoscale organization of SHROOM2 at tight junctions and its interactions with the cytoskeleton at resolutions below the diffraction limit .

• Multi-omics approaches: Coupling SHROOM2 immunoprecipitation using biotin-conjugated antibodies with mass spectrometry can identify novel interaction partners and post-translational modifications.

• Live-cell imaging techniques: Developing cell-permeable biotin-conjugated SHROOM2 antibody fragments for real-time visualization of protein dynamics during morphogenesis or cell migration.

• Spatial transcriptomics: Combining biotin-conjugated SHROOM2 immunodetection with in situ RNA sequencing to correlate protein localization with local transcriptional profiles.

• Microfluidic platforms: Integrating antibody-based detection in organ-on-chip models to study SHROOM2 function in physiologically relevant microenvironments.

• CRISPR-Cas9 screens with immunophenotyping: Using biotin-conjugated SHROOM2 antibodies to assess phenotypic consequences of genome-wide CRISPR screens affecting SHROOM2 pathways.

These integrated approaches can significantly advance understanding of SHROOM2's role in development, physiology, and disease processes .