SHROOM2 Antibody, HRP conjugated

What is SHROOM2 protein and what cellular functions does it serve?

SHROOM2 (also known as Protein APXL) is a cytoskeletal protein that functions as a key mediator of the RhoA-ROCK pathway, regulating cell motility and actin cytoskeleton organization. In endothelial cells, SHROOM2 is involved in morphology changes during cell spreading processes. Within the retinal pigment epithelium, SHROOM2 has been demonstrated to regulate the biogenesis of melanosomes and promote their association with the apical cell surface by inducing gamma-tubulin redistribution . Recent research has also identified an unanticipated role for SHROOM2 in suppressing epithelial-to-mesenchymal transition (EMT) and tumor metastasis, suggesting its potential function as a tumor suppressor in certain cancers like nasopharyngeal carcinoma .

How does HRP conjugation benefit SHROOM2 antibody applications?

HRP (horseradish peroxidase) conjugation provides significant advantages for detection sensitivity in immunoassay applications. The enzyme catalyzes a reaction with chemiluminescent substrates to produce a luminescent signal that can be detected and quantified. This conjugation eliminates the need for secondary antibody incubation steps, streamlining experimental protocols and reducing background noise. HRP-conjugated antibodies offer unparalleled sensitivity for detecting low abundance proteins, making them particularly valuable for SHROOM2 detection in samples where expression may be limited or downregulated . The HRP moiety enables direct visualization in applications such as ELISA, with signal amplification properties that enhance detection of even minute quantities of the target protein.

What are the recommended storage conditions for SHROOM2 antibody, HRP conjugated?

For optimal preservation of activity, SHROOM2 antibody, HRP conjugated should be stored at -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles should be strictly avoided as they can compromise antibody functionality and HRP enzymatic activity. Most commercial preparations are supplied in a stabilizing buffer containing 50% glycerol and 0.01M PBS (pH 7.4) with preservatives such as 0.03% Proclin 300 to maintain antibody integrity during storage . When working with the antibody, it is advisable to aliquot the stock solution into smaller volumes to minimize freeze-thaw cycles. For short-term storage during experimental procedures, the antibody can be temporarily kept at 4°C, but should be returned to -20°C or -80°C for long-term storage to preserve the activity of both the antibody and the HRP conjugate.

How is the specificity of SHROOM2 antibody, HRP conjugated verified?

Specificity verification for SHROOM2 antibody, HRP conjugated involves multiple validation approaches to ensure reliable target recognition. Commercial antibodies are typically raised against specific immunogens, such as recombinant Human Protein Shroom2 fragments (213-405AA) . Validation typically includes positive control testing with human samples known to express SHROOM2, western blotting to confirm binding to proteins of the expected molecular weight, and immunohistochemistry to evaluate tissue distribution patterns. Cross-reactivity testing against related proteins helps establish specificity boundaries. For research applications, researchers should review the antibody datasheet for validation data and may need to perform additional validation specific to their experimental system, including antibody titration to determine optimal working dilutions and negative control experiments using samples where SHROOM2 is absent or knocked down.

What is the optimal protocol for using SHROOM2 antibody, HRP conjugated in ELISA applications?

For ELISA applications using SHROOM2 antibody, HRP conjugated, the following methodological approach is recommended:

• Coating: Coat the ELISA plate wells with the capture antigen or antibody (depending on whether direct or sandwich ELISA) at 1-10 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C.

• Blocking: Block non-specific binding sites with 1-5% BSA or non-fat milk in PBS-T (PBS + 0.05% Tween-20) for 1-2 hours at room temperature.

• Sample addition: Add samples containing target SHROOM2 protein and incubate for 1-2 hours at room temperature.

• Antibody application: Apply the SHROOM2 antibody, HRP conjugated at a dilution of 1:500-1:1000 as recommended for ELISA applications . Incubate for 1 hour at room temperature.

• Washing: Perform stringent washing (4-6 times) with PBS-T between each step to reduce background.

• Detection: Add HRP substrate such as TMB (3,3',5,5'-Tetramethylbenzidine) and incubate for 15-30 minutes protected from light.

• Reaction stopping: Stop the reaction with 2N H₂SO₄ or other appropriate stop solution.

• Measurement: Measure absorbance at 450 nm with a reference wavelength of 620 nm.

For optimal results, each new lot of antibody should be titrated to determine the exact optimal dilution that provides maximum specific signal with minimal background. Including positive and negative controls is essential for proper interpretation of results.

How can SHROOM2 antibody, HRP conjugated be utilized to study the RhoA-ROCK pathway?

SHROOM2 antibody, HRP conjugated can be strategically employed to investigate the RhoA-ROCK pathway through several methodological approaches:

• Co-immunoprecipitation studies: Use the antibody to pull down SHROOM2 and analyze associated proteins within the RhoA-ROCK pathway through subsequent western blotting.

• Pathway activation monitoring: Compare SHROOM2 expression levels before and after RhoA-ROCK pathway stimulation or inhibition using ELISA. The HRP conjugation allows direct quantification of expression changes.

• Inhibitor studies: Combine ROCK inhibitors like Y-27632 with SHROOM2 detection to examine synergistic effects, as research indicates that "SHROOM2 and ROCK work synergistically rather than epistatically" .

• Cytoskeletal reorganization analysis: Following treatment with cytoskeleton-disrupting agents, quantify SHROOM2 levels to understand its role in actin reorganization during cell spreading and cytoskeletal remodeling.

• Tumor metastasis models: When studying cancer models, particularly nasopharyngeal carcinoma where SHROOM2 downregulation has been observed , the antibody can be used to correlate protein expression with metastatic potential.

It's important to note that while SHROOM2 is involved in the RhoA-ROCK pathway for stress fiber formation and focal adhesion, it also has ROCK-independent functions in suppressing epithelial-to-mesenchymal transition, making it a multifaceted research target requiring careful experimental design .

What controls should be included when using SHROOM2 antibody, HRP conjugated in experimental workflows?

A robust experimental design using SHROOM2 antibody, HRP conjugated should incorporate the following essential controls:

• Positive control: Include samples known to express SHROOM2, such as normal nasopharyngeal epithelial (NPE) cell lines, which demonstrate higher SHROOM2 expression compared to nasopharyngeal carcinoma (NPC) cell lines .

• Negative control: Utilize samples with confirmed absence of SHROOM2 expression, or employ SHROOM2 knockdown models through siRNA or CRISPR-Cas9 approaches.

• Isotype control: Include a matched isotype IgG (from rabbit for the antibodies described in the search results) conjugated to HRP to assess non-specific binding.

• Dilution series control: Prepare a standard curve using recombinant SHROOM2 protein at known concentrations to validate quantification accuracy.

• Secondary antibody only control: For comparison experiments with non-conjugated primary antibodies, include wells with only secondary antibody to evaluate background.

• Substrate-only control: Include wells with HRP substrate but no antibody to establish baseline readings for the detection system.

• Blocking peptide control: Use the immunizing peptide (aa 213-405 or relevant region) to confirm antibody specificity through competitive binding.

• Cross-reactivity controls: If studying related SHROOM family proteins, include samples expressing SHROOM1, SHROOM3, or SHROOM4 to verify antibody specificity within the protein family.

These controls help validate results, troubleshoot experimental issues, and provide confidence in data interpretation, particularly when studying the complex roles of SHROOM2 in cellular processes.

How can SHROOM2 antibody, HRP conjugated be used to investigate tumor suppressor activity in cancer research?

Recent findings indicating SHROOM2's role in tumor suppression can be investigated using HRP-conjugated antibodies through several sophisticated methodological approaches:

• Tissue microarray analysis: Quantify SHROOM2 expression levels across tumor progression stages using standardized immunohistochemistry protocols. Evidence shows that "SHROOM2 expression was significantly downregulated in human NPC compared to non-cancerous nasopharyngeal tissues" and "the expression of SHROOM2 in metastatic NPC was even lower than in the primary tumors" .

• EMT marker correlation studies: Employ multiplex ELISA to simultaneously quantify SHROOM2 and epithelial-to-mesenchymal transition markers. Research indicates that "Depletion of SHROOM2 in nasopharyngeal carcinoma (NPC) cells enhances mesenchymal characteristics and reduces epithelial markers, concomitant with increased motility" .

• ROCK pathway inhibition experiments: Combine SHROOM2 detection with ROCK inhibitors (such as Y-27632) to distinguish between ROCK-dependent and ROCK-independent functions of SHROOM2 in tumor suppression.

• Migration and invasion assays: Correlate SHROOM2 expression levels with cell migration capacity using wound healing or transwell assays, as "combination of ROCK inhibition and SHROOM2 depletion resulted in the most robust increases in cell migration and invasion" .

• Clinical sample analysis: Develop prognostic scoring systems based on SHROOM2 expression patterns in patient samples using the following methodology:

This scoring system, similar to that used in the cited research , can help establish SHROOM2 as a prognostic biomarker for cancer progression.

What methodologies can address the challenge of detecting low SHROOM2 expression in cancer tissues?

Detecting low abundance SHROOM2 in cancer tissues, where expression is often downregulated, requires specialized methodological approaches:

• Signal amplification techniques: Utilize tyramide signal amplification (TSA) in conjunction with HRP-conjugated SHROOM2 antibodies to exponentially increase detection sensitivity by depositing multiple tyramide-fluorophore or tyramide-chromogen molecules at the antibody binding site.

• Optimized antigen retrieval: Implement comprehensive antigen retrieval optimization matrices testing multiple pH conditions (citrate buffer pH 6.0, EDTA pH 8.0, and Tris-EDTA pH 9.0) combined with varying heat application methods (microwave, pressure cooker, or water bath) to maximize epitope accessibility.

• Prolonged antibody incubation: For tissues with low SHROOM2 expression, extend primary antibody incubation to 18-24 hours at 4°C using optimized antibody concentration (typically between 1:250-1:500 dilution).

• Enhanced chemiluminescence systems: Pair HRP-conjugated antibodies with highly sensitive detection substrates like Azure Radiance chemiluminescent reagents that are specifically optimized for low abundance proteins.

• Microfluidic immunoassay techniques: Employ microfluidic platforms that use minimal sample volumes and create more favorable kinetics for antibody-antigen interactions, enhancing detection of sparse SHROOM2 protein.

• Multiplexed detection systems: Combine SHROOM2 antibody with antibodies against related pathway proteins to create a detection signature that compensates for low individual protein expression.

• Image analysis algorithms: Implement computational image analysis using machine learning algorithms specifically trained to detect subtle SHROOM2 staining patterns against tissue background.

When implementing these approaches, it's critical to maintain appropriate controls, especially when pushing detection limits, to distinguish true positive signals from potential artifacts.

How can conflicting data regarding SHROOM2 expression patterns across different cancer types be reconciled?

Reconciliation of conflicting SHROOM2 expression data across cancer types requires systematic methodological approaches:

• Standardized antibody validation: Implement rigorous antibody validation across cancer studies using the same HRP-conjugated SHROOM2 antibody with consistent epitope targeting (e.g., aa 213-405) . Document specificity through western blotting, immunoprecipitation, and peptide blocking experiments.

• Isoform-specific detection: Design detection strategies accounting for potential SHROOM2 isoforms or post-translational modifications that might be differentially expressed across cancer types. This requires:

  • Epitope mapping to determine antibody recognition sites

  • Isoform-specific primers for complementary qPCR validation

  • Mass spectrometry confirmation of protein variants

• Contextual analysis framework: Develop a standardized analysis framework that contextualizes SHROOM2 expression within:

  • Tumor microenvironment characteristics

  • Patient demographic and clinical parameters

  • Treatment history

  • RhoA-ROCK pathway activation status

• Meta-analysis methodology: Employ statistical approaches that account for:

  • Interlaboratory variability

  • Differences in tissue processing

  • Variations in detection methods

  • Antibody lot-to-lot variations

• Correlation with functional readouts: Establish standard functional assays measuring:

  • Cell migration capacity

  • Stress fiber formation

  • EMT marker expression

  • Melanosome distribution (in appropriate cell types)

By implementing these methodological approaches, researchers can better understand whether conflicting SHROOM2 expression patterns represent true biological differences between cancer types or are artifacts of methodological inconsistencies, providing a more coherent picture of SHROOM2's role across different cancers.

What are the most effective strategies for optimizing signal-to-noise ratio when using SHROOM2 antibody, HRP conjugated?

Optimizing signal-to-noise ratio with SHROOM2 antibody, HRP conjugated requires systematic methodology refinement:

• Antibody titration matrix: Perform comprehensive titration experiments testing dilutions ranging from 1:250 to 1:2000, extending beyond the manufacturer's recommended range of 1:500-1:1000 . Plot signal-to-noise ratio at each dilution to identify the optimal concentration.

• Blocking optimization: Test multiple blocking agents systematically:

  • 1-5% BSA in PBS-T

  • 1-5% non-fat milk in PBS-T

  • Commercial blocking reagents

  • Species-matched normal serum (2-10%)

• Washing protocol refinement: Implement stringent washing protocols with increasing stringency:

  • Standard: 3 x 5 minutes with PBS-T (0.05% Tween-20)

  • Intermediate: 5 x 5 minutes with PBS-T (0.1% Tween-20)

  • Stringent: 5 x 10 minutes with PBS-T (0.1% Tween-20) + 350mM NaCl

• Substrate selection optimization: Compare multiple HRP substrates for optimal performance with SHROOM2 detection:

  • TMB for colorimetric detection

  • Enhanced chemiluminescent substrates like Azure Radiance

  • Fluorescent HRP substrates for alternative detection methods

• Sample preparation refinement: Optimize protein extraction methods to maximize SHROOM2 yield while minimizing interfering substances:

  • Test multiple lysis buffers (RIPA, NP-40, Triton X-100)

  • Evaluate protease inhibitor cocktail compositions

  • Compare sonication vs. homogenization methods

• Cross-adsorption of antibody: Consider using cross-adsorbed antibody preparations to reduce non-specific binding if background remains problematic.

• Data analysis algorithms: Implement background subtraction algorithms specific to the detection method used, particularly important for densitometric analysis of western blots or quantitative ELISA data interpretation.

These methodical optimization steps should be performed sequentially, documenting results at each stage to establish an optimized protocol specific to the experimental conditions and biological system being investigated.

How should researchers address potential cross-reactivity between SHROOM2 antibody and other SHROOM family proteins?

Addressing potential cross-reactivity between SHROOM family proteins requires a systematic validation approach:

• Epitope sequence analysis: Perform computational alignment of the immunogen sequence (aa 213-405 or other relevant epitopes) across all SHROOM family members (SHROOM1-4) to identify regions of homology that might contribute to cross-reactivity.

• Recombinant protein panel testing: Develop a validation panel using:

  • Purified recombinant SHROOM1-4 proteins

  • Synthetic peptides corresponding to potential cross-reactive epitopes

  • Chimeric proteins with swapped domains between SHROOM family members

• Knockout/knockdown validation matrix: Implement a comprehensive validation using:

  • CRISPR-Cas9 SHROOM2 knockout cell lines as negative controls

  • siRNA knockdown of each SHROOM family member individually

  • Overexpression systems for each SHROOM protein

• Competitive binding assays: Perform pre-absorption experiments where the antibody is pre-incubated with:

  • Immunizing peptide (positive control for blocking)

  • Homologous peptides from other SHROOM family members

  • Unrelated control peptides

• Multi-antibody validation approach: Compare results using multiple antibodies targeting different SHROOM2 epitopes:

  • Central region antibodies (aa 599-625)

  • N-terminal antibodies

  • C-terminal antibodies

• Species cross-reactivity assessment: Evaluate antibody performance across species with varying degrees of SHROOM2 homology to identify species-specific recognition patterns that may inform epitope specificity.

• Documentation standards: Establish rigorous documentation of all validation experiments, including:

  • Full western blot images showing all bands

  • Complete peptide competition results

  • Raw data from cross-reactivity experiments

This comprehensive validation approach not only addresses cross-reactivity concerns but also builds confidence in experimental results involving SHROOM2 detection in complex biological systems.

What methodological approaches can determine if SHROOM2 antibody, HRP conjugated maintains functionality in different experimental conditions?

Assessing functional stability of SHROOM2 antibody, HRP conjugated across experimental conditions requires systematic methodology:

• Temperature stability assessment: Evaluate antibody performance after exposure to different temperatures using standardized ELISA:

  • Storage at recommended temperature (-20°C or -80°C)

  • Room temperature exposure (1, 4, 8, 24 hours)

  • Elevated temperature stress (37°C for 1, 4, 8, 24 hours)

  • Multiple freeze-thaw cycles (1-5 cycles)

• Buffer compatibility matrix: Test antibody function in different buffer systems relevant to various applications:

  • Standard PBS (pH 7.4)

  • Tris-buffered saline (pH 7.6)

  • Carbonate buffer (pH 9.6) for ELISA coating

  • Cell lysis buffers (RIPA, NP-40)

  • Antigen retrieval solutions (citrate, EDTA)

• Detergent tolerance profiling: Establish tolerance to commonly used detergents:

  • Tween-20 (0.05-0.5%)

  • Triton X-100 (0.1-1%)

  • SDS (0.01-0.1%)

  • Digitonin (0.01-0.1%)

• pH stability assessment: Characterize antibody performance across pH range:

  • Acidic conditions (pH 4.0-6.0)

  • Neutral conditions (pH 6.5-7.5)

  • Basic conditions (pH 8.0-10.0)

• Storage time evaluation: Establish long-term stability profile through testing at:

  • Initial time point (baseline)

  • 1 month, 3 months, 6 months, 12 months intervals

• Accelerated aging studies: Perform accelerated aging tests using elevated temperatures to predict long-term stability.

• HRP activity verification: Separate antibody binding assessment from HRP functionality using:

  • Direct ELISA to measure binding capacity

  • Colorimetric HRP substrate assays to specifically measure enzyme activity

• Data analysis methodology: Implement statistical approaches to determine:

  • Significant deviations from baseline performance

  • Establish acceptance criteria for each application

  • Develop predictive models for stability

These methodological approaches enable researchers to establish validated protocols for handling SHROOM2 antibody, HRP conjugated across diverse experimental conditions while maintaining confidence in results.

How might SHROOM2 antibody, HRP conjugated facilitate investigation of SHROOM2's role in neuroscience research?

SHROOM2 antibody, HRP conjugated offers significant potential for advancing neuroscience research through the following methodological approaches:

• Neural development studies: Track SHROOM2 expression during neuronal differentiation and migration using immunocytochemistry with HRP-conjugated antibodies to visualize protein localization at different developmental stages.

• Cytoskeletal dynamics in neurons: Investigate SHROOM2's involvement in neuronal cytoskeletal organization during axon growth and dendrite formation through:

  • Quantitative immunocytochemistry with HRP detection

  • Co-localization studies with cytoskeletal markers

  • Comparative expression analysis before and after treatment with cytoskeletal disrupting agents

• Synapse formation and plasticity: Examine SHROOM2's potential role in synaptic structure through quantitative approaches:

  • Synaptic fractionation followed by ELISA using HRP-conjugated antibodies

  • Comparison of SHROOM2 levels in models of synaptic plasticity

  • Correlation of SHROOM2 expression with electrophysiological measurements

• Neurodevelopmental disorder models: Investigate SHROOM2 expression in models of neurodevelopmental disorders using standardized ELISA protocols with HRP-conjugated antibodies to establish potential biomarker applications.

• Neural tissue-specific expression mapping: Develop comprehensive neural expression atlases using:

  • Brain region-specific quantification

  • Cell type-specific analysis through co-labeling strategies

  • Developmental stage comparisons

• Neuronal RhoA-ROCK pathway analysis: Extend the established role of SHROOM2 in the RhoA-ROCK pathway to neural-specific contexts, examining how this pathway regulates neuronal morphology and function.

The designation of SHROOM2 as relevant to neuroscience research suggests untapped potential for understanding its functions in neural tissues that can be effectively explored using the high sensitivity afforded by HRP-conjugated antibodies.

What emerging methodologies could enhance the utility of SHROOM2 antibody, HRP conjugated in studying actin cytoskeleton organization?

Emerging methodologies for studying SHROOM2's role in actin cytoskeleton organization using HRP-conjugated antibodies include:

• Super-resolution microscopy integration: Combine HRP-mediated tyramide signal amplification with super-resolution techniques (STORM, PALM, STED) to:

  • Visualize SHROOM2 localization at nanoscale resolution

  • Map precise spatial relationships between SHROOM2 and actin structures

  • Track dynamic changes during cell spreading events

• Live-cell compatible proximity labeling: Adapt HRP-based proximity labeling techniques:

  • Develop cell-permeable HRP substrates for intracellular labeling

  • Create temporal maps of SHROOM2 interactions with cytoskeletal elements

  • Quantify interaction dynamics during morphological changes

• Automated high-content screening platforms: Implement advanced imaging workflows:

  • Develop machine learning algorithms for automated cytoskeletal feature recognition

  • Establish multiplexed readouts combining SHROOM2 detection with actin visualization

  • Create standardized morphometric analysis parameters for quantitative comparison

• Microfluidic force application systems: Integrate SHROOM2 detection with mechanical stimulation:

  • Apply defined mechanical forces to cells while monitoring SHROOM2 distribution

  • Correlate cytoskeletal reorganization with SHROOM2 recruitment patterns

  • Measure force-dependent changes in SHROOM2-mediated pathways

• Biomimetic substrates with tunable stiffness: Study how substrate properties affect SHROOM2 function:

  • Compare SHROOM2 distribution on substrates of varying rigidity

  • Correlate mechanical properties with cytoskeletal organization patterns

  • Develop 3D models that better recapitulate in vivo environments

• Optogenetic SHROOM2 regulation: Combine light-controlled protein modules with SHROOM2:

  • Create optically-triggered SHROOM2 recruitment or degradation systems

  • Observe real-time cytoskeletal responses to SHROOM2 spatial modulation

  • Map temporal dynamics of downstream cytoskeletal changes

These emerging methodologies would significantly enhance our understanding of SHROOM2's role in "endothelial cell morphology changes during cell spreading" and potentially reveal new therapeutic targets for pathologies involving cytoskeletal dysregulation.

How can SHROOM2 antibody, HRP conjugated contribute to understanding the mechanisms of melanosome biogenesis in relation to SHROOM2 function?

SHROOM2 antibody, HRP conjugated can significantly advance understanding of melanosome biogenesis through sophisticated methodological approaches:

• Quantitative co-localization studies: Develop dual-labeling protocols combining:

  • HRP-labeled SHROOM2 antibody detection with chromogenic substrates

  • Fluorescent markers for melanosome stages (PMEL17, TYRP1, RAB27A)

  • Quantitative co-localization analysis using specialized software

• Temporal mapping of biogenesis: Implement pulse-chase experiments:

  • Track newly synthesized melanosomes using time-resolved labeling

  • Correlate SHROOM2 recruitment with specific stages of melanosome maturation

  • Quantify distribution patterns during "association with the apical cell surface"

• High-resolution ultrastructural analysis: Combine HRP detection with electron microscopy:

  • Utilize HRP for electron-dense precipitate formation visible by EM

  • Map SHROOM2 localization at ultrastructural level around melanosomes

  • Correlate with "gamma-tubulin redistribution" using dual-labeling approaches

• Gamma-tubulin interaction studies: Investigate the mechanism behind SHROOM2-induced "gamma-tubulin redistribution" :

  • Develop co-immunoprecipitation protocols optimized for cytoskeletal proteins

  • Establish ELISA-based interaction assays using HRP-conjugated antibodies

  • Create in vitro reconstitution systems to test direct interaction capabilities

• Melanosome trafficking analysis: Track melanosome movement in relation to SHROOM2:

  • Time-lapse microscopy with cells expressing fluorescent melanosome markers

  • Quantitative tracking of movement parameters before and after SHROOM2 manipulation

  • Correlation with cytoskeletal organization patterns

• Cross-comparison with retinal disease models: Investigate SHROOM2 function in:

  • Retinal pigment epithelium (RPE) cell culture models

  • Patient-derived RPE cells with pigmentation disorders

  • Animal models of retinal pigmentation defects

• SHROOM2 domain-function mapping: Determine which SHROOM2 domains mediate melanosome interactions:

  • Express truncated SHROOM2 constructs lacking specific domains

  • Quantify effects on melanosome distribution using HRP-labeled antibodies

  • Establish structure-function relationships for melanosome association

These methodological approaches could significantly advance our understanding of SHROOM2's role in melanosome biogenesis and potentially reveal new insights into pigmentation disorders and retinal pathologies.

What are the most important methodological considerations for researchers beginning work with SHROOM2 antibody, HRP conjugated?

Researchers initiating studies with SHROOM2 antibody, HRP conjugated should prioritize these methodological considerations:

• Comprehensive validation: Before beginning experimental work, validate the antibody in your specific experimental system:

  • Confirm target specificity through western blotting

  • Verify appropriate dilution ranges beyond manufacturer recommendations of 1:500-1:1000

  • Test in positive control samples known to express SHROOM2, such as normal nasopharyngeal epithelial cells

• Protocol optimization hierarchy: Establish optimized protocols in this sequence:

  • Sample preparation (fixation, permeabilization, antigen retrieval)

  • Blocking conditions (agent, concentration, duration)

  • Antibody concentration and incubation parameters

  • Washing stringency

  • Detection system optimization

• Context-specific controls: Implement controls appropriate to the biological question:

  • For tumor suppressor studies: paired normal/tumor tissue controls

  • For cytoskeletal studies: positive controls with defined cytoskeletal states

  • For developmental studies: stage-specific reference samples

• Storage and handling precision: Follow strict storage guidelines at -20°C or -80°C , with:

  • Aliquoting to prevent freeze-thaw cycles

  • Careful temperature monitoring during shipping and handling

  • Regular activity testing of stored antibody

• Application-appropriate analysis: Select analysis methods based on experimental goals:

  • Quantitative analysis for expression level comparisons

  • High-resolution imaging for localization studies

  • Multiplexed approaches for pathway analysis

• Cross-laboratory standardization: Establish consistent methodologies across research teams:

  • Standardized positive controls

  • Shared analysis algorithms

  • Common reporting formats for results

By adhering to these methodological fundamentals, researchers can establish reliable, reproducible protocols for SHROOM2 research that will facilitate meaningful comparisons across studies and advance understanding of this multifunctional protein.

How can researchers integrate SHROOM2 antibody, HRP conjugated into multi-parameter studies of cytoskeletal regulation?

Integrating SHROOM2 antibody, HRP conjugated into multi-parameter cytoskeletal studies requires sophisticated methodological approaches:

• Multiplexed detection systems: Develop protocols combining:

  • HRP-conjugated SHROOM2 antibody with chromogenic detection

  • Fluorescently-labeled antibodies against cytoskeletal components

  • Sequential detection with multiple HRP-conjugated antibodies using tyramide signal amplification with spectral unmixing

• Pathway activation signature mapping: Create standardized readout panels:

  • Phosphorylation status of RhoA-ROCK pathway components

  • Actin polymerization state markers

  • Focal adhesion complex components

  • SHROOM2 expression and localization

• Multi-scale imaging approach: Implement hierarchical imaging strategies:

  • Low-magnification screening for population-level patterns

  • High-resolution imaging of regions of interest

  • Super-resolution techniques for molecular-scale organization

• Integrated mechanical and biochemical analysis: Combine:

  • Atomic force microscopy for cell stiffness measurements

  • Traction force microscopy for cell-generated forces

  • SHROOM2 expression quantification in the same cells

• Temporal dynamics integration: Develop time-resolved approaches:

  • Live-cell compatible proximal labeling techniques

  • Fixed-time-point series with synchronized populations

  • Computational models predicting SHROOM2 dynamics based on endpoint measurements

• Data integration frameworks: Establish computational pipelines for:

  • Multi-parameter correlation analysis

  • Principal component analysis of cytoskeletal features

  • Machine learning classification of cellular states based on SHROOM2 and cytoskeletal parameters