ARHGEF19 Antibody

What is ARHGEF19 and what is its function in cellular processes?

ARHGEF19 is a member of the RhoGEF family that functions as a guanyl-nucleotide exchange factor, facilitating the activation of Rho GTPases by promoting the exchange of GDP for GTP. This protein contains two key functional domains: the Dbl homology (DH) domain and the pleckstrin homology (PH) domain, which are essential for its guanine nucleotide exchange activity . These domains are characteristic of the Ephexin-like protein family .

At the cellular level, ARHGEF19 participates in various physiological processes including survival, development, differentiation, and motility through regulation of cytoskeletal dynamics and cell signaling pathways . In developmental contexts, ARHGEF19 has been demonstrated to act upstream of or within epidermis development and neural tube closure processes, as evidenced by studies in model organisms like zebrafish .

The protein is expressed in specific tissues, with notable expression in the post-vent region in zebrafish models, suggesting tissue-specific functions during development . Recent research has also identified elevated ARHGEF19 expression in human breast cancer, indicating a potential role in cancer progression or metastasis that warrants further investigation .

What types of ARHGEF19 antibodies are currently available for research applications?

Several types of ARHGEF19 antibodies are available for research applications, each with specific characteristics and applications:

Most commercially available antibodies are raised in rabbits and generated against synthetic peptides derived from specific regions of human ARHGEF19. The typical molecular weight detected is approximately 89 kDa, corresponding to the full-length protein . While most antibodies demonstrate specific reactivity to human ARHGEF19, cross-reactivity with other species may vary depending on sequence homology and should be validated experimentally.

How are ARHGEF19 antibodies validated for research applications?

Validation of ARHGEF19 antibodies involves multiple approaches to ensure specific and reliable detection of the target protein:

• Immunohistochemistry (IHC) validation typically uses positive control tissues known to express ARHGEF19, such as human lung carcinoma tissue, which has been documented to show positive staining with validated antibodies .

• Western blot validation confirms detection of a band at the expected molecular weight of approximately 89 kDa, which corresponds to the calculated molecular weight of ARHGEF19 (802 amino acids) .

• Antibody specificity is verified through affinity purification methods, with many commercial antibodies being purified using affinity chromatography with epitope-specific immunogens to ensure selective binding to ARHGEF19 .

• Optimization of dilution ratios is performed for each application, with manufacturers generally recommending specific dilution ranges: 1:50-1:300 for IHC, 1:500-1:2000 for Western blot, and 1:20000 for ELISA applications .

• Some manufacturers provide validation images showing antibody performance in different applications, such as immunohistochemistry of paraffin-embedded human lung carcinoma tissue, allowing researchers to assess whether the antibody is suitable for their specific experimental needs .

What are the optimal conditions for using ARHGEF19 antibodies in immunohistochemistry?

Achieving optimal results with ARHGEF19 antibodies in immunohistochemistry requires careful attention to several methodological parameters:

• Tissue preparation: Formalin-fixed, paraffin-embedded (FFPE) human tissue sections are commonly used, as demonstrated in validation studies using human lung carcinoma tissue . Fixation time should be standardized to preserve epitope integrity while maintaining tissue morphology.

• Antigen retrieval: Heat-induced epitope retrieval in appropriate buffers is typically necessary to unmask antigens that may have been cross-linked during fixation. This step is crucial for accessing the ARHGEF19 epitopes.

• Blocking conditions: To reduce background staining, sections should be blocked with appropriate blocking solutions. The specific composition may vary depending on the detection system used.

• Primary antibody dilution: The recommended dilutions for ARHGEF19 antibodies in IHC applications typically range from 1:50 to 1:300, depending on the specific antibody . For example, antibody A14835-1 is recommended at 1:100-1:300 dilution for IHC applications .

• Incubation conditions: Overnight incubation at 4°C generally yields better results than shorter incubations at room temperature, especially for antibodies with moderate affinity.

• Detection system: A compatible secondary antibody (typically goat anti-rabbit for most ARHGEF19 antibodies) and appropriate visualization system should be selected based on the primary antibody host species.

• Controls: Include positive controls (tissues known to express ARHGEF19) and negative controls (primary antibody omitted) to validate staining specificity.

Optimization of these parameters should be performed for each new antibody and tissue type to achieve specific and reproducible results.

What are the recommended protocols for Western blot applications with ARHGEF19 antibodies?

For optimal Western blot results with ARHGEF19 antibodies, consider the following protocol recommendations:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors to extract ARHGEF19 protein efficiently while preventing degradation. RIPA buffer or NP-40 based lysis buffers are commonly effective.

• Protein loading: Load 20-40 μg of total protein per lane for cell lysates or 50-100 μg for tissue lysates to ensure adequate detection of ARHGEF19.

• Gel selection: Use 8-10% polyacrylamide gels for optimal resolution of ARHGEF19, which has a molecular weight of approximately 89 kDa .

• Transfer conditions: Transfer to PVDF membranes (preferred over nitrocellulose for high molecular weight proteins) at appropriate voltage and time.

• Blocking: Block membranes with 5% non-fat dry milk or 5% BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute ARHGEF19 antibodies according to manufacturer recommendations, typically in the range of 1:500 to 1:2000 for Western blot applications . Incubate overnight at 4°C with gentle agitation.

• Washing: Perform thorough washing steps with TBST to remove unbound primary antibody.

• Secondary antibody: Use an appropriate HRP-conjugated secondary antibody (anti-rabbit for most ARHGEF19 antibodies) at the recommended dilution.

• Detection: Employ enhanced chemiluminescence (ECL) detection methods appropriate for the expected signal intensity.

If non-specific bands appear, optimization steps may include increasing blocking time, decreasing antibody concentration, or adding 0.1-0.5% Tween-20 to the antibody dilution buffer to reduce non-specific binding.

How should researchers design experiments to study ARHGEF19 function in development and disease?

Designing experiments to investigate ARHGEF19 function requires careful consideration of model systems, functional assays, and detection methods:

• Model system selection: Choose appropriate models based on ARHGEF19 expression and research question:

  • For developmental studies: Zebrafish models are valuable as ARHGEF19 has been shown to function in epidermis development and neural tube closure in this organism .

  • For cancer research: Human cell lines with documented ARHGEF19 expression, particularly breast cancer lines, are appropriate given the reported high expression in this cancer type .

• Functional approaches:

  • Gain-of-function: Overexpression of wild-type or tagged ARHGEF19 constructs to assess effects on Rho GTPase activation, cytoskeletal reorganization, and cell behavior.

  • Loss-of-function: siRNA knockdown, shRNA, or CRISPR/Cas9 knockout of ARHGEF19 to determine the consequences of its absence.

• Domain analysis: Create constructs with mutations or deletions in the DH or PH domains to investigate domain-specific functions in Rho GTPase activation and cellular processes .

• Interaction studies: Employ co-immunoprecipitation or proximity ligation assays to identify ARHGEF19 binding partners and regulatory proteins.

• Functional readouts: As ARHGEF19 is a RhoGEF, appropriate readouts include:

  • Rho GTPase activation assays (pull-down of active GTP-bound Rho)

  • Cytoskeletal remodeling assessment (F-actin organization)

  • Cell migration and invasion assays

  • Developmental phenotype analysis in model organisms

• Validation of antibody specificity: Confirm antibody specificity in your experimental system, particularly when using knockdown/knockout approaches as controls.

This multi-faceted approach allows for comprehensive characterization of ARHGEF19 function in normal and pathological contexts.

How can researchers investigate post-translational modifications of ARHGEF19?

Studying post-translational modifications (PTMs) of ARHGEF19 requires specialized approaches:

• Immunoprecipitation followed by targeted analysis:

  • Use validated ARHGEF19 antibodies to immunoprecipitate the protein from cell or tissue lysates

  • Probe the immunoprecipitate with antibodies against specific PTMs (e.g., anti-phosphotyrosine, anti-phosphoserine)

  • Alternatively, analyze immunoprecipitated ARHGEF19 by mass spectrometry to identify multiple PTMs simultaneously

• Phos-tag SDS-PAGE: This technique incorporates Phos-tag molecules into polyacrylamide gels, retarding the migration of phosphorylated proteins. When combined with standard ARHGEF19 antibodies in Western blotting, this allows detection of phosphorylated forms as higher-molecular-weight bands.

• Site-directed mutagenesis: After identifying potential modification sites, generate mutants (e.g., phospho-null or phospho-mimetic) to assess the functional significance of specific modifications.

• In vitro kinase assays: Identify kinases that may phosphorylate ARHGEF19 through candidate approaches or kinase screening platforms.

• Domain-specific analysis: Given the critical functional domains in ARHGEF19 (DH and PH domains), investigate how PTMs within these regions affect GEF activity and protein-protein interactions .

This area remains relatively unexplored for ARHGEF19 and presents opportunities for novel discoveries about its regulation and function in different cellular contexts.

What strategies can be used to investigate ARHGEF19's role in cancer progression?

Investigating ARHGEF19's role in cancer progression, particularly breast cancer where it shows high expression , requires multifaceted approaches:

• Expression profiling:

  • Use validated ARHGEF19 antibodies for IHC analysis of tumor microarrays containing multiple cancer types and matched normal tissues

  • Quantify expression levels and correlate with clinical parameters (stage, grade, survival)

  • Compare expression patterns in primary tumors versus metastatic lesions

• Functional studies in cancer models:

  • Overexpression studies in cancer cell lines to assess effects on proliferation, migration, and invasion

  • Knockdown/knockout approaches to determine whether ARHGEF19 is necessary for malignant phenotypes

  • Mouse xenograft models to evaluate the impact on tumor growth and metastasis in vivo

• Mechanistic investigations:

  • Assess ARHGEF19's effect on Rho GTPase activation in cancer cells

  • Determine downstream signaling pathways affected by ARHGEF19 modulation

  • Investigate interactions with known oncogenes or tumor suppressors

• Therapeutic targeting potential:

  • Develop strategies to inhibit ARHGEF19 function (small molecules targeting GEF activity or protein-protein interactions)

  • Evaluate combination approaches with standard chemotherapeutics

  • Assess ARHGEF19 as a biomarker for patient stratification or treatment response

• Antibody-based detection methods:

  • Use immunofluorescence to determine subcellular localization changes in cancer cells

  • Employ multiplexed antibody approaches to correlate ARHGEF19 expression with other cancer biomarkers

  • Develop standardized IHC protocols for consistent detection in patient samples

These approaches can provide valuable insights into the potential roles of ARHGEF19 in cancer initiation, progression, and metastasis, potentially identifying new therapeutic targets or biomarkers.

How can ARHGEF19 antibodies be used in conjunction with live cell imaging techniques?

Combining ARHGEF19 antibodies with live cell imaging techniques requires creative experimental design:

• Antibody-based approaches for fixed cell imaging:

  • Immunofluorescence using validated ARHGEF19 antibodies can provide high-resolution images of endogenous protein localization

  • Multiplexed imaging with markers of cytoskeletal structures, adhesion complexes, or active Rho GTPases can reveal functional associations

  • Super-resolution microscopy techniques (STED, STORM, PALM) can provide nanoscale localization information

• Complementary approaches for live cell imaging:

  • Fluorescent protein fusions: Generate ARHGEF19-GFP (or other fluorescent protein) fusions for direct visualization in living cells

  • Validate fusion protein functionality and localization using antibodies against endogenous ARHGEF19

  • Use FRET-based biosensors to monitor ARHGEF19 activity or interactions in real-time

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence imaging of ARHGEF19 (using antibodies or fluorescent proteins) with electron microscopy

  • This approach allows visualization of ARHGEF19 in the context of ultrastructural features

• Optogenetic approaches:

  • Develop light-controllable ARHGEF19 variants to manipulate its activity with temporal and spatial precision

  • Use antibodies to validate expression and localization of these engineered proteins

• Validation strategies:

  • Confirm specificity of fluorescent signals using ARHGEF19 knockdown/knockout controls

  • Perform parallel experiments with multiple antibodies targeting different epitopes

  • Compare live and fixed cell imaging results to ensure consistency

These approaches enable dynamic studies of ARHGEF19 function and provide spatial information that is crucial for understanding its role in processes like cell migration, division, and response to extracellular signals.

What are common problems with ARHGEF19 detection in Western blots and how can they be resolved?

Researchers may encounter several challenges when detecting ARHGEF19 in Western blots, with the following solutions recommended:

• Weak or absent signal:

  • Increase protein loading (start with 50-100 μg of total protein)

  • Optimize primary antibody concentration (try a range from 1:500 to 1:2000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced sensitivity detection reagents

  • Ensure transfer efficiency for high molecular weight proteins (~89 kDa) by adding SDS to transfer buffer or increasing transfer time

• Multiple bands or non-specific signals:

  • Increase blocking stringency (try 5% BSA instead of milk, or commercial blocking reagents)

  • Reduce primary antibody concentration

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

  • Increase washing steps (number and duration)

  • Perform peptide competition assay to identify specific bands

  • Consider the possibility of splice variants or post-translational modifications

• High background:

  • Use freshly prepared buffers

  • Ensure adequate blocking (try extending blocking time to 2 hours)

  • Dilute primary antibody in fresh blocking buffer

  • Check secondary antibody compatibility and specificity

  • Clean the membrane thoroughly between antibody incubations

• Inconsistent results between experiments:

  • Standardize sample preparation protocols

  • Use fresh protease inhibitors in lysis buffers

  • Implement consistent handling of primary antibody (avoid freeze-thaw cycles)

  • Consider preparing single-use aliquots of antibody

  • Include positive control samples in each experiment

• Validation controls:

  • Include lysates from cells with ARHGEF19 knockdown/knockout as negative controls

  • Use recombinant ARHGEF19 protein as a positive control if available

  • Consider using multiple antibodies targeting different epitopes to confirm results

Following these troubleshooting steps should help achieve specific and reproducible detection of ARHGEF19 in Western blot applications.

How can researchers optimize immunohistochemistry protocols for ARHGEF19 detection in different tissue types?

Optimizing immunohistochemistry protocols for ARHGEF19 detection across different tissue types requires systematic adjustment of several parameters:

• Tissue preparation considerations:

  • Fixation: Standardize fixation time (typically 24-48 hours in 10% neutral buffered formalin)

  • Processing: Ensure consistent tissue processing and embedding

  • Section thickness: Use 4-5 μm sections for optimal antibody penetration and morphology

• Antigen retrieval optimization:

  • Test multiple methods: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) vs. EDTA buffer (pH 9.0)

  • Optimization of retrieval time: Test ranges from 10-30 minutes

  • Pressure vs. non-pressure systems: Compare results with pressure cooker, microwave, or water bath methods

• Blocking and antibody conditions:

  • Blocking agents: Compare normal serum, BSA, commercial blocking solutions

  • Primary antibody dilution: Test serial dilutions (1:50, 1:100, 1:200, 1:300) to determine optimal concentration

  • Incubation time/temperature: Compare room temperature (1-2 hours) vs. 4°C (overnight)

  • Diluent composition: Test addition of 0.1-0.3% Triton X-100 for improved penetration

• Detection system selection:

  • Compare polymer-based detection systems vs. traditional avidin-biotin methods

  • Evaluate signal amplification approaches for tissues with low ARHGEF19 expression

  • Adjust chromogen development time based on expression levels

• Tissue-specific adaptations:

  • For tissues with high endogenous peroxidase (e.g., liver, kidney): Extend peroxidase blocking step

  • For fatty tissues: Consider extended deparaffinization steps

  • For tissues with high background: Implement additional blocking steps

• Validation approaches:

  • Always include positive control tissues (e.g., human lung carcinoma)

  • Include negative controls (primary antibody omitted)

  • Compare staining patterns with mRNA expression data where available

Systematic optimization and documentation of these parameters will help establish reliable protocols for ARHGEF19 detection across different tissue types.

What strategies should be employed when antibody validation results are inconsistent?

When faced with inconsistent antibody validation results, researchers should implement a systematic troubleshooting approach:

• Antibody characterization:

  • Review the immunogen information: Different antibodies target different epitopes of ARHGEF19, which may affect detection in certain applications or conditions

  • Compare polyclonal vs. recombinant antibodies: Recombinant antibodies may offer greater batch-to-batch consistency

  • Check for potential cross-reactivity with related proteins, particularly other RhoGEF family members

• Technical validation approaches:

  • Genetic validation: Test antibody in samples with ARHGEF19 knockdown/knockout

  • Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Tagged protein expression: Compare antibody detection with tagged ARHGEF19 detected via the tag

• Application-specific considerations:

  • For Western blotting: Optimize protein extraction, gel percentage, transfer conditions

  • For IHC/IF: Test multiple fixation and antigen retrieval methods

  • For ELISA: Evaluate coating conditions, blocking agents, and detection systems

• Experimental variables assessment:

  • Sample preparation: Ensure consistent handling and processing

  • Reagent quality: Check for degradation or contamination of buffers and antibodies

  • Protocol standardization: Document all experimental conditions meticulously

• Methodological triangulation:

  • Use multiple antibodies targeting different epitopes of ARHGEF19

  • Employ complementary techniques (e.g., mRNA detection, MS-based proteomics)

  • Compare results across different biological systems

• Validation reporting:

  • Document all validation experiments thoroughly

  • Report both successful and unsuccessful conditions

  • Specify exact antibody used (catalog number, lot) in publications

This systematic approach helps resolve inconsistencies and ensures reliable antibody-based detection of ARHGEF19 across different experimental systems.

How might ARHGEF19 antibodies contribute to understanding developmental disorders?

ARHGEF19 antibodies can play a crucial role in investigating developmental disorders, building on evidence of its involvement in key developmental processes:

• Developmental pathway analysis:

  • ARHGEF19 has been implicated in epidermis development and neural tube closure in zebrafish models

  • Antibody-based detection can map ARHGEF19 expression patterns during critical developmental windows

  • Co-localization with developmental markers can reveal associations with specific morphogenetic processes

• Comparative studies in model organisms:

  • Use validated antibodies to compare ARHGEF19 expression and localization across species

  • Investigate evolutionary conservation of function through comparative analysis

  • Correlate expression patterns with developmental phenotypes in genetic models

• Human developmental disorder investigations:

  • Explore ARHGEF19 expression in relevant human tissues from patients with developmental disorders

  • Analyze potential mutations or expression changes in conditions affecting epidermis development or neural tube closure

  • Develop diagnostic applications based on ARHGEF19 detection

• Mechanistic studies:

  • Investigate ARHGEF19 interactions with developmental signaling pathways (Wnt, Notch, BMP)

  • Assess how ARHGEF19-mediated Rho GTPase activation influences cell behavior during development

  • Examine potential developmental roles of ARHGEF19 domains (DH, PH, SH3) through structure-function analysis

• Therapeutic implications:

  • Explore ARHGEF19 as a potential therapeutic target for developmental disorders

  • Use antibodies to validate target engagement of experimental therapeutics

  • Develop screening assays for compounds that modulate ARHGEF19 function

These approaches could yield valuable insights into the molecular mechanisms underlying developmental disorders and potentially identify new diagnostic or therapeutic strategies.

What are emerging applications of ARHGEF19 antibodies in cancer research?

ARHGEF19 antibodies are enabling several innovative approaches in cancer research, particularly given the evidence of high expression in breast cancer :

• Biomarker development:

  • Standardized IHC protocols using validated ARHGEF19 antibodies for patient stratification

  • Correlation of expression levels with clinical parameters and treatment response

  • Multiplexed detection with other cancer biomarkers to develop comprehensive profiles

• Metastasis research:

  • Investigation of ARHGEF19 expression in primary tumors versus metastatic lesions

  • Analysis of its role in epithelial-mesenchymal transition and cell invasion

  • Correlation with metastatic potential and circulating tumor cell characteristics

• Targeted therapy approaches:

  • Development of antibody-drug conjugates targeting ARHGEF19

  • Screening for small molecule inhibitors of ARHGEF19 GEF activity

  • Use of antibodies to validate target engagement in preclinical studies

• Tumor microenvironment interactions:

  • Analysis of ARHGEF19 expression in stromal cells versus cancer cells

  • Investigation of its role in cancer-associated fibroblast activation

  • Exploration of potential immune modulatory functions

• Resistance mechanisms:

  • Evaluation of ARHGEF19 expression changes in treatment-resistant tumors

  • Analysis of its role in cancer stem cell maintenance

  • Investigation of compensatory signaling mechanisms

• Personalized medicine applications:

  • Development of companion diagnostics based on ARHGEF19 detection

  • Exploration of synthetic lethality approaches with ARHGEF19 inhibition

  • Patient selection for clinical trials based on ARHGEF19 expression profiles

These emerging applications highlight the potential significance of ARHGEF19 as both a biomarker and therapeutic target in cancer research.

How can multi-omics approaches be integrated with ARHGEF19 antibody-based studies?

Integrating multi-omics approaches with ARHGEF19 antibody-based studies can provide comprehensive insights into its function and regulation:

• Proteogenomic integration:

  • Correlate ARHGEF19 protein levels (detected by antibodies) with corresponding mRNA expression

  • Identify potential post-transcriptional regulatory mechanisms

  • Investigate the impact of genetic alterations on protein expression and function

• Phosphoproteomics approaches:

  • Combine ARHGEF19 immunoprecipitation with mass spectrometry to identify phosphorylation sites

  • Correlate phosphorylation status with activation state and functional outcomes

  • Develop phospho-specific antibodies for key regulatory sites

• Interactome analysis:

  • Use antibody-based pull-down combined with mass spectrometry to identify ARHGEF19 binding partners

  • Validate key interactions using proximity ligation assays or co-immunoprecipitation

  • Map interaction networks in different cellular contexts and disease states

• Spatial multi-omics:

  • Combine antibody-based detection of ARHGEF19 with spatial transcriptomics

  • Correlate protein localization with local gene expression patterns

  • Develop multiplexed imaging approaches to simultaneously detect multiple proteins

• Functional genomics integration:

  • Correlate CRISPR screening phenotypes with ARHGEF19 expression patterns

  • Identify synthetic lethal interactions through combined genetic and antibody-based approaches

  • Validate genetic dependencies using protein detection methods

• Data integration frameworks:

  • Develop computational approaches to integrate antibody-based protein data with other omics data types

  • Apply machine learning algorithms to identify patterns and correlations

  • Create predictive models of ARHGEF19 function and regulation

This integrated approach allows researchers to move beyond simple detection of ARHGEF19 to a systems-level understanding of its roles in normal physiology and disease.