SHROOM1 Antibody

What are the confirmed applications for SHROOM1 antibodies in laboratory research?

SHROOM1 antibodies have been validated for multiple research applications with varying degrees of optimization. Current evidence supports their use in:

• Western Blot (WB): Most commercially available SHROOM1 antibodies are validated for WB with recommended dilutions ranging from 1:500 to 1:3000

• Immunoprecipitation (IP): Successfully demonstrated with MCF-7 cells using 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Enzyme-Linked Immunosorbent Assay (ELISA): Validated in multiple antibody formulations

When designing experiments, researchers should note that very few SHROOM1 antibodies have been validated for immunohistochemistry (IHC) or immunofluorescence (ICC-IF) applications, though some manufacturers do offer products claiming these capabilities .

What is the observed molecular weight of SHROOM1 in experimental conditions?

SHROOM1 consistently appears at a higher molecular weight than its calculated value in experimental conditions. While the calculated molecular weight based on amino acid sequence is approximately 91 kDa, the observed molecular weight in SDS-PAGE and Western blot analysis is typically 100-130 kDa . This discrepancy likely reflects post-translational modifications. Researchers should anticipate this migration pattern when interpreting Western blot results and consider including appropriate positive controls such as lysates from MCF7 or A375 cells, which have been confirmed to express detectable levels of endogenous SHROOM1 .

What cell lines are recommended as positive controls for SHROOM1 expression?

For validation of SHROOM1 antibodies and experimental design, the following cell lines have been confirmed to express detectable levels of endogenous SHROOM1:

• MCF7 (human breast adenocarcinoma cell line)

• A375 (human malignant melanoma cell line)

These cell lines should be considered when establishing experimental protocols requiring positive controls. When using SHROOM1 antibodies for the first time in a particular cell type or tissue, researchers should include these positive control lysates to validate antibody performance and establish appropriate exposure conditions.

What are the recommended storage conditions for maintaining SHROOM1 antibody activity?

To maintain optimal activity of SHROOM1 antibodies, consistent storage protocols are essential:

• Temperature: Store at -20°C for up to one year from receipt date

• Aliquoting: While some products specify that aliquoting is unnecessary for -20°C storage , it is generally recommended to prepare small aliquots to avoid repeated freeze-thaw cycles that may compromise antibody performance

• Thawing: Thaw antibodies slowly on ice rather than at room temperature to preserve binding capacity

• Buffer conditions: Most commercial SHROOM1 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

Researchers should verify the specific storage recommendations for their particular antibody formulation, as minor variations exist between manufacturers.

How should SHROOM1 antibody dilution optimization be approached for Western blot applications?

For optimal Western blot results with SHROOM1 antibodies, a systematic dilution optimization approach is recommended:

• Start with the manufacturer's suggested dilution range (typically 1:500-1:3000 for SHROOM1 antibodies)

• Perform a dilution series experiment using consistent protein loading (recommended 20-40 μg total protein per lane)

• Include positive control lysates (MCF7 or A375 cells) alongside experimental samples

• Evaluate signal-to-noise ratio at each dilution point

• Consider secondary antibody optimization in parallel if background issues persist

• Document optimal conditions for reproducibility across experiments

Remember that sample-dependent factors may necessitate adjustments to standard protocols, and manufacturers acknowledge that "optimal dilutions/concentrations should be determined by the end user" .

How does SHROOM1 antibody selection differ for investigations into neural tube morphogenesis versus pulmonary arterial research?

When investigating SHROOM1 in different tissue contexts, antibody selection should consider target epitope location and tissue-specific protein conformations:

For neural tube morphogenesis research:

• Select antibodies targeting the PDZ domain or regions involved in actin binding, as SHROOM1 functions as a PDZ domain-containing actin-binding protein required for neural tube morphogenesis

• Consider antibodies validated in neurological tissues or neuronal cell lines

• Verify cross-reactivity with your model organism, noting that sequence homology between human and mouse/rat SHROOM1 is approximately 51%

For pulmonary arterial research:

• SHROOM1 expression is significantly decreased in mouse and human models of pulmonary arterial hypertension

• Select antibodies validated for detecting expression level changes rather than just presence/absence

• Consider antibodies tested in pulmonary arterial smooth muscle cells

• Evaluate whether the antibody recognizes post-translational modifications that may be tissue-specific

In both contexts, researchers should perform preliminary validation experiments to confirm antibody specificity and sensitivity in their specific tissue or cell type of interest.

What considerations are important when using SHROOM1 antibodies in CRISPR-Cas9 genome editing experiments?

The relationship between SHROOM1 and CRISPR-Cas9 genome editing efficiency presents unique experimental considerations:

• Baseline measurement: Establish baseline SHROOM1 expression levels before knockdown experiments, as SHROOM1 suppression improves CRISPR-mediated precision genome editing

• Antibody validation for knockdown verification:

  • Select antibodies with demonstrated sensitivity to detect at least 50% reduction in protein levels

  • Consider using multiple antibodies targeting different epitopes to verify knockdown efficiency

  • Quantify knockdown efficiency using densitometric analysis of Western blots

• Time-course monitoring:

  • SHROOM1 knockdown-enhanced HDR efficiency is time-dependent, with optimal effects observed 24-28 days after cell line construction

  • Design experiments to monitor SHROOM1 levels throughout the editing timeline

• Controls for mechanistic studies:

  • Include appropriate controls when investigating whether SHROOM1 knockdown enhances HDR through an HDR-dependent mechanism

  • Research indicates SHROOM1 knockdown-enhanced editing can be counteracted by HDR inhibitors (e.g., YU238259) but not by NHEJ inhibitors (e.g., Scr7)

These considerations ensure accurate interpretation of experimental results when investigating SHROOM1's role in genome editing efficiency.

What methodological approaches can resolve discrepancies in SHROOM1 antibody specificity across different experimental systems?

When encountering inconsistent SHROOM1 antibody performance across experimental systems, implement the following methodological approaches:

• Multiple antibody validation strategy:

  • Test multiple antibodies targeting different epitopes of SHROOM1

  • Compare polyclonal and monoclonal antibodies, as each has distinct advantages for different applications

  • Document epitope locations for each antibody to identify potential region-specific accessibility issues

• Knockout/knockdown validation:

  • Validate antibody specificity using SHROOM1 knockout or knockdown controls

  • Research has demonstrated successful SHROOM1 knockout in HEK293T cells using SpCas9 and two sgRNAs targeting exon 4

  • Verify complete protein loss in knockout models through Western blot analysis

• Cross-reactivity assessment:

  • Evaluate cross-reactivity with other SHROOM family members (SHROOM2-4)

  • Note that SHROOM family proteins share conserved domains (ASD domains), which may lead to cross-reactivity

  • SHROOM1, like other family members (except SHROOM4), contains both ASD1 and ASD2 domains

• Sample preparation optimization:

  • Optimize protein extraction methods for different tissue types

  • Test different lysis buffers to ensure complete solubilization of membrane-associated SHROOM1

  • Consider native versus denaturing conditions depending on epitope accessibility

Implementing these approaches systematically can resolve discrepancies and establish reliable protocols for specific experimental systems.

How can SHROOM1 antibodies be effectively used to investigate interactions with ROCK1 and the cytoskeletal network?

To investigate SHROOM1 interactions with ROCK1 and the cytoskeletal network:

• Co-immunoprecipitation optimization:

  • While no direct SHROOM1-ROCK1 interaction has been documented in the provided materials, studies of SHROOM2 provide a methodological template

  • SHROOM2 research demonstrates successful co-immunoprecipitation using anti-Myc agarose beads at 4°C for 2 hours

  • Similar approaches could be adapted for SHROOM1 studies, focusing on the ASD domains which may mediate interactions with ROCK1

• Cytoskeletal co-localization studies:

  • SHROOM1 may be involved in the assembly of microtubule arrays during cell elongation

  • Design double immunofluorescence experiments using SHROOM1 antibodies alongside markers for:

    • Actin filaments (phalloidin staining)

    • Microtubules (α-tubulin antibodies)

    • Centrosomes (γ-tubulin antibodies)

  • Ectopic expression of SHROOM1 has been shown to alter γ-tubulin distribution in epithelial cells

• Functional domain analysis:

  • SHROOM1 contains PDZ and ASD domains important for its function

  • Design experiments to correlate antibody epitope location with functional domains

  • Consider using domain-specific antibodies to investigate region-specific interactions

These methodological approaches can help elucidate SHROOM1's role in cytoskeletal organization and its potential interaction partners.

What techniques can address the challenges of detecting low SHROOM1 expression in pulmonary arterial hypertension models?

Detecting reduced SHROOM1 expression in pulmonary arterial hypertension models requires specialized techniques:

• Signal amplification methods:

  • Implement tyramide signal amplification (TSA) for immunohistochemistry

  • Use highly sensitive chemiluminescent substrates for Western blot detection

  • Consider proximity ligation assay (PLA) for detecting SHROOM1 interactions with enhanced sensitivity

• Enrichment protocols:

  • Perform subcellular fractionation to concentrate SHROOM1 in cytoskeletal fractions

  • Implement immunoprecipitation before Western blot analysis to concentrate target protein

  • Use laser capture microdissection to isolate specific pulmonary arterial smooth muscle cells before analysis

• Quantitative comparison approaches:

  • Employ quantitative Western blot using internal loading controls

  • Normalize SHROOM1 expression to multiple housekeeping proteins

  • Implement digital PCR for transcript quantification in parallel with protein detection

• Alternative detection methods:

  • Consider mass spectrometry-based proteomics for unbiased detection and quantification

  • Implement RNA-scope for sensitive mRNA detection in tissue sections

  • Use CRISPR-Cas9 knock-in of epitope tags for enhanced detection sensitivity

These techniques can overcome the challenges associated with detecting diminished SHROOM1 expression in disease models where expression is significantly decreased .

What are the most common causes of non-specific binding with SHROOM1 antibodies and how can they be mitigated?

Non-specific binding with SHROOM1 antibodies can arise from several sources with specific mitigation strategies:

For persistent non-specific binding issues, consider switching to monoclonal antibodies or testing antibodies from different manufacturers that target distinct epitopes.

How can researchers validate SHROOM1 antibody specificity in the context of genetic manipulation experiments?

A comprehensive validation strategy for SHROOM1 antibodies in genetic manipulation experiments should include:

• Knockout verification:

  • Generate SHROOM1 knockout controls using CRISPR-Cas9 targeting exon 4

  • Verify knockout by genomic sequencing to confirm frameshift mutations

  • Demonstrate complete absence of target band in Western blot analysis

  • Documented knockout approach achieved 815 bp deletion and +1 bp frameshift in HEK293T cells

• Knockdown gradient analysis:

  • Perform siRNA-mediated knockdown with varying concentrations

  • Establish correlation between knockdown efficiency and antibody signal reduction

  • Use multiple siRNAs targeting different regions of SHROOM1 mRNA

• Overexpression controls:

  • Generate tagged SHROOM1 overexpression constructs

  • Compare antibody detection with tag-specific antibodies

  • Verify size shift with epitope-tagged constructs

• Cross-species validation:

  • Test antibody performance across species with known sequence differences

  • Note that sequence homology between human and mouse/rat SHROOM1 is approximately 51%

  • Use samples from relevant model organisms to confirm specificity

• Epitope mapping:

  • Perform Western blot with truncated SHROOM1 constructs to verify epitope location

  • Use peptide competition assays with the immunogen sequence

  • Consider the impact of post-translational modifications on epitope recognition

These methodological approaches establish robust validation of antibody specificity critical for accurate interpretation of genetic manipulation experiments.