RHOJ Antibody

What is RHOJ and why is it significant in research?

RHOJ (ras homolog family member J) is a member of the Rho protein family with a canonical length of 214 amino acid residues and molecular mass of 23.8 kDa in humans . It is predominantly localized in the cell membrane and is notably expressed in endothelial cells across various tissues including brain, heart, lung, and liver . Its significance stems from its involvement in cytoskeleton organization and angiogenesis . RHOJ has been implicated in cancer progression, particularly in melanoma invasion and glioblastoma, making it an important target for oncology research .

What are the common applications for RHOJ antibodies in research?

RHOJ antibodies are primarily utilized for:

• Western blotting (WB) to detect and quantify RHOJ protein expression

• Immunohistochemistry (IHC) to visualize RHOJ in tissue sections

• Immunocytochemistry (ICC) and immunofluorescence (IF) to determine subcellular localization

• Enzyme-linked immunosorbent assay (ELISA) for quantitative measurement

• Immunoprecipitation (IP) to isolate RHOJ and its binding partners

Research contexts frequently include angiogenesis studies, cancer progression models, and cytoskeletal dynamics investigations .

How can I distinguish between different RHOJ isoforms using antibodies?

Alternative splicing generates two distinct RHOJ isoforms . To distinguish between these isoforms:

• Select antibodies raised against unique epitopes specific to each isoform

• Use Western blotting with high-resolution gels (10-12% polyacrylamide) to separate the isoforms based on subtle size differences

• Perform preliminary validation using recombinant proteins of each isoform as positive controls

• Consider using RT-PCR with isoform-specific primers in parallel to confirm antibody specificity

• When possible, use knockout/knockdown models as negative controls to verify antibody specificity for each isoform

What are the critical controls needed when using RHOJ antibodies in experimental studies?

A robust experimental design with RHOJ antibodies requires:

Positive controls:

• Cell lines known to express high levels of RHOJ (endothelial cells are ideal)

• Recombinant RHOJ protein

• RHOJ-overexpressing cells (via transfection or viral transduction)

Negative controls:

• RHOJ-depleted cells using validated siRNA or shRNA

• Non-expressing cell lines

• Isotype controls to assess non-specific binding

• Secondary antibody-only controls to evaluate background signal

Normalization controls:

• Loading controls for Western blots (tubulin, actin)

• Reference transcripts for corresponding qPCR validation

• Internal tissue controls when performing IHC

Additionally, include solvent controls when treating samples with compounds that might affect RHOJ expression or function .

How should I optimize RHOJ antibody concentration for immunohistochemistry of glioblastoma tissues?

For optimal RHOJ antibody staining in glioblastoma tissues:

• Begin with a titration experiment using a dilution series (typically 1:50 to 1:500) of the primary antibody

• Use known positive controls like endothelial cells or validated RHOJ-positive glioblastoma samples

• Include negative controls (secondary antibody only, isotype control)

• Optimize antigen retrieval methods—RHOJ detection in glioblastoma sections typically requires heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0)

• Determine optimal incubation conditions (duration and temperature)

• Validate specificity using dual staining with endothelial markers like CD31 to confirm RHOJ's endothelial expression pattern

• Standardize to a 1:100 dilution for most applications with overnight incubation at 4°C, as demonstrated in successful glioblastoma studies

What considerations should be made when selecting between monoclonal and polyclonal RHOJ antibodies?

Monoclonal RHOJ Antibodies:

• Advantages: High specificity for a single epitope, reduced batch-to-batch variation, excellent for distinguishing between closely related proteins

• Limitations: May lose reactivity if the single epitope is masked, denatured, or modified

• Best applications: Quantitative assays, therapeutic development, studies requiring high reproducibility

Polyclonal RHOJ Antibodies:

• Advantages: Recognize multiple epitopes, more robust against protein modifications, generally provide stronger signals

• Limitations: Potential cross-reactivity, batch-to-batch variation

• Best applications: Initial screening, detection of low-abundance proteins, applications where protein may be partially denatured

For critical research focusing on specific RHOJ conformations or post-translational modifications (like palmitoylation), monoclonal antibodies targeted to those specific regions are recommended .

How can RHOJ antibodies be used to investigate the role of RHOJ in tumor angiogenesis?

To investigate RHOJ's role in tumor angiogenesis:

• Tissue expression analysis:

  • Use immunohistochemistry with RHOJ antibodies on tumor tissue sections

  • Co-stain with endothelial markers (CD31) to correlate RHOJ expression with vascular density

  • Compare expression levels between different tumor grades

• Functional studies:

  • Establish knockdown/knockout models using shRNA/siRNA against RHOJ

  • Use RHOJ antibodies to confirm knockdown efficiency by Western blot

  • Assess vascular parameters using tube formation assays and in vivo models

• Signaling pathway analysis:

  • Investigate RHOJ interaction with angiogenic pathways like VEGFR2 signaling

  • Use phospho-specific antibodies alongside RHOJ antibodies to evaluate pathway activation

  • Perform co-immunoprecipitation with RHOJ antibodies to identify binding partners

• In vivo models:

  • Generate xenograft models with RHOJ-depleted cells

  • Use RHOJ antibodies for IHC analysis of tumor vasculature

  • Correlate RHOJ expression with metastatic potential and tumor invasiveness

This integrated approach has revealed RHOJ's critical role in glioblastoma angiogenesis via the JNK/VEGFR2 signaling pathway .

What techniques can be used to study RHOJ-mediated cytoskeletal reorganization in melanoma cells?

To investigate RHOJ's role in cytoskeletal reorganization:

• Actin cytoskeleton visualization:

  • Transfect cells with RHOJ expression vectors or deplete RHOJ using siRNA/shRNA

  • Verify expression/depletion using RHOJ antibodies via Western blot

  • Stain fixed cells with rhodamine phalloidin to visualize F-actin

  • Analyze cell morphology parameters (length, shape) using ImageJ software

• Migration and invasion assays:

  • Conduct Boyden chamber assays with RHOJ-modified cells

  • Perform collagen I invasion assays to assess ECM penetration ability

  • Document scratch assay healing in the presence of mitomycin C (to exclude proliferation effects)

• Downstream effector analysis:

  • Use RHOJ antibodies alongside antibodies against downstream proteins (cofilin, LIMK2, PAK1)

  • Assess phosphorylation states of these effectors via Western blotting

  • Correlate RHOJ expression with effector activation

• Live cell imaging:

  • Generate fluorescently-tagged RHOJ constructs

  • Monitor cytoskeletal dynamics in real-time using confocal microscopy

  • Correlate RHOJ localization with areas of active cytoskeletal remodeling

This methodological approach has demonstrated that RHOJ regulates melanoma cell migration and invasion by controlling actin cytoskeletal dynamics through the PAK-LIMK-cofilin pathway .

How can I design experiments to investigate RHOJ's role in glioblastoma progression using antibody-based approaches?

To study RHOJ in glioblastoma progression:

• Expression profiling across tumor grades:

  • Perform IHC with RHOJ antibodies on different grade glioma tissues

  • Use 1:100 dilution of anti-RHOJ antibody (Abnova H00057381-M01)

  • Quantify expression levels across tumor grades to establish correlation with malignancy

• Functional validation:

  • Generate stable RHOJ knockdown in glioblastoma cell lines using lentiviral shRNA

  • Confirm knockdown efficiency using Western blot with RHOJ antibodies

  • Assess effects on proliferation, migration, and invasion in vitro

• Signaling pathway investigation:

  • Use co-immunoprecipitation with RHOJ antibodies to identify binding partners

  • Perform Western blotting to analyze downstream signaling (JNK/VEGFR2 pathway)

  • Use pharmacological inhibitors of identified pathways to confirm specificity

• In vivo validation:

  • Establish xenograft models using RHOJ-depleted cells

  • Monitor tumor growth and perform endpoint analysis

  • Conduct IHC on tumor sections using RHOJ and CD31 antibodies to assess vascular density

  • Analyze lymph node metastasis using human-specific markers

This comprehensive approach has successfully demonstrated that RHOJ is a novel target for glioblastoma progression and invasion, with potential therapeutic implications .

How do I address false positive results when using RHOJ antibodies in immunohistochemistry?

To minimize false positives in RHOJ IHC:

• Antibody validation:

  • Verify antibody specificity using Western blot on tissues/cells with known RHOJ expression

  • Include RHOJ knockdown samples as negative controls

  • Test with recombinant RHOJ protein for positive confirmation

• Protocol optimization:

  • Titrate primary antibody concentration (typically 1:100 dilution shows specific staining)

  • Optimize blocking conditions (5% BSA or 10% normal serum from secondary antibody host)

  • Include endogenous peroxidase blocking step for HRP-based detection

  • Test multiple antigen retrieval methods to determine optimal conditions

• Controls implementation:

  • Include isotype control antibodies at the same concentration

  • Perform secondary-only control staining

  • Use non-relevant tissues known to be RHOJ-negative

  • Include internal positive control (endothelial cells) in each section

• Signal validation:

  • Confirm expression pattern (membrane localization) is consistent with RHOJ biology

  • Validate with dual immunofluorescence using known RHOJ-associated markers

  • Compare staining pattern with published literature

What strategies should I employ when RHOJ antibody shows inconsistent results across different experimental batches?

To address batch-to-batch inconsistency:

• Standardize reagents and protocols:

  • Use the same antibody lot when possible (record lot numbers)

  • Prepare fresh buffers for each experiment

  • Implement detailed SOPs with precise timing and temperature control

  • Standardize sample preparation methods

• Implement design of experiments (DoE) approach:

  • Utilize blocking in experimental design to minimize run-to-run variance

  • Consider randomized complete block design (RCBD) for large sample sets

  • Systematically test critical parameters that might affect antibody performance

• Technical validation:

  • Include positive controls (recombinant protein, high-expressing cells) in every experiment

  • Normalize results to consistent internal standards

  • Consider multiple detection methods (e.g., validate WB results with ELISA)

• Statistical approaches:

  • Increase biological and technical replicates

  • Use appropriate statistical tests to account for batch effects

  • Consider mixed-effects models for data with known batch variation

• Alternative approaches:

  • Validate key findings with a second RHOJ antibody targeting a different epitope

  • Complement antibody-based detection with mRNA analysis (qPCR)

  • Consider using reporter systems for functional studies

How can I differentiate between specific RHOJ signal and cross-reactivity with other Rho family GTPases?

To distinguish RHOJ-specific signals from cross-reactivity:

• Antibody selection:

  • Choose antibodies targeting unique regions of RHOJ that differ from other Rho GTPases

  • Review the immunogen sequence used to generate the antibody

  • Select monoclonal antibodies for higher specificity when distinguishing between closely related proteins

• Validation with genetic models:

  • Use RHOJ knockdown/knockout models as negative controls

  • Include overexpression models of related Rho GTPases (particularly RHOG) to test for cross-reactivity

  • Perform parallel experiments with antibodies against other Rho family members

• Biochemical validation:

  • Perform peptide competition assays using RHOJ-specific peptides

  • Use recombinant RHOJ and related GTPases (RHOG, CDC42) for Western blot validation

  • Consider 2D gel electrophoresis to separate closely related Rho proteins

• Orthogonal validation:

  • Complement protein detection with mRNA analysis using isoform-specific primers

  • Use activity-based assays specific to RHOJ function

  • When possible, utilize mass spectrometry to confirm antibody specificity

RHOJ and RHOG share sequence homology, making careful validation particularly important when working in systems where both proteins may be expressed .

How can RHOJ antibodies be utilized in studying the role of RHOJ in endothelial cell-specific functions?

To investigate RHOJ in endothelial cell functions:

• Expression profiling:

  • Use RHOJ antibodies for Western blot and IHC analysis across different endothelial cell types

  • Compare expression levels between arterial, venous, and lymphatic endothelial cells

  • Assess regulation under different stimuli (growth factors, hypoxia, inflammatory cytokines)

• Promoter studies:

  • Combine RHOJ antibody protein detection with promoter analysis

  • Use luciferase reporter assays with RHOJ promoter constructs

  • Correlate protein expression with transcriptional regulation by factors like ERG

• Functional analysis:

  • Generate endothelial-specific RHOJ knockdown/overexpression models

  • Use adenoviral vectors expressing GFP-RhoJ for localization studies

  • Assess effects on tube formation, migration, and vessel stabilization

• In vivo vascular morphogenesis:

  • Develop endothelial-specific RHOJ knockout mouse models

  • Use RHOJ antibodies for IHC analysis of vascular development

  • Correlate RHOJ expression with vessel maturation markers

These approaches have established RHOJ as an endothelial cell-restricted Rho GTPase that mediates vascular morphogenesis and is regulated by the transcription factor ERG .

What methodological approaches can determine the relationship between RHOJ post-translational modifications and protein function?

To study RHOJ post-translational modifications (PTMs):

• PTM-specific detection:

  • Use antibodies that specifically recognize palmitoylated RHOJ

  • Employ biochemical techniques like acyl-biotin exchange (ABE) to detect palmitoylation

  • Use mass spectrometry to identify and map other PTMs

• Functional correlation:

  • Generate RHOJ mutants lacking specific modification sites

  • Verify expression using standard RHOJ antibodies

  • Compare localization and function between wild-type and mutant proteins

  • Use live cell imaging to track modified vs. unmodified RHOJ

• Pathway analysis:

  • Investigate how PTMs affect RHOJ interaction with downstream effectors

  • Use co-immunoprecipitation with RHOJ antibodies followed by Western blot for binding partners

  • Compare signaling pathway activation between modified and unmodified RHOJ

• Pharmacological manipulation:

  • Use inhibitors of palmitoylation (2-bromopalmitate) or other PTM-modifying enzymes

  • Assess effects on RHOJ localization, activity, and downstream signaling

  • Validate using RHOJ antibodies in combination with subcellular fractionation

This multifaceted approach can reveal how PTMs like palmitoylation regulate RHOJ membrane localization and subsequent signaling activities critical for its biological functions .

What are the most reliable commercial RHOJ antibodies for specific research applications?

Based on published research and validation data, the following RHOJ antibodies have demonstrated reliability in specific applications:

• For immunohistochemistry (IHC) in glioblastoma and cancer tissues:

  • Abnova RHOJ Antibody (H00057381-M01) at 1:100 dilution

  • Thermo Fisher Scientific RhoJ Antibody (validated for IHC-p)

• For Western blotting:

  • Aviva Systems Biology RHOJ antibody (ARP57431_P050) for human, mouse, and rabbit samples

  • Novus Biologicals RhoJ Antibody (1E4) with validation across multiple applications

• For immunofluorescence:

  • BosterBio Anti-RHOJ Mouse Monoclonal Antibody for human, mouse, and rat samples

  • Santa Cruz Biotechnology Rho J (FF-19) Antibody with validated IF applications

• For multi-application research:

  • Atlas Antibodies Anti-RHOJ Antibody with validation in IHC, ICC-IF, and WB

  • United States Biological Rabbit Anti-RHOJ antibodies for WB, ELISA, and IHC applications