ARHGEF18 Antibody

What is the molecular weight of ARHGEF18 and why might I observe variations in Western blot results?

ARHGEF18 has a calculated molecular weight of approximately 131 kDa, though the observed molecular weight typically ranges between 120-130 kDa in Western blot applications . This variation could be attributed to:

• Post-translational modifications affecting protein migration

• The existence of multiple isoforms, particularly the recently discovered "LOCGEF" isoforms (such as the 1361-residue X3 isoform) prevalent in certain cell types like eosinophils

• Cell or tissue-specific expression patterns of different isoforms

• Sample preparation conditions affecting protein folding

When troubleshooting unexpected band patterns, consider validating with multiple antibodies recognizing different epitopes and comparing results with published literature on tissue-specific expression patterns .

What antibody isotypes and hosts are available for ARHGEF18 detection?

Most commercially available ARHGEF18 antibodies are:

Rabbit polyclonal antibodies are most commonly used due to their ability to recognize multiple epitopes, enhancing detection sensitivity. For more specific applications requiring detection of particular domains or isoforms, various region-specific antibodies are available targeting N-terminal, C-terminal, or center regions of the protein .

What are the recommended storage conditions for maintaining ARHGEF18 antibody activity?

For optimal preservation of antibody activity:

• Store at -20°C in aliquots to avoid repeated freeze-thaw cycles

• Most ARHGEF18 antibodies remain stable for one year after shipment when stored properly

• Commercial preparations typically contain stabilizers such as 50% glycerol and 0.02% sodium azide in PBS (pH 7.3)

• Small volume aliquots (20μl) may contain 0.1% BSA as an additional stabilizer

• Aliquoting is generally unnecessary for -20°C storage according to manufacturer recommendations

Proper storage ensures consistent experimental results by preventing antibody degradation and maintaining specificity for target recognition .

What are the recommended dilution ratios for different applications of ARHGEF18 antibodies?

Optimal dilution ratios vary by application technique:

It is essential to titrate the antibody in your specific experimental system to obtain optimal signal-to-noise ratios. Sample-dependent optimization may be necessary, particularly for detecting differential expression across tissues or treatment conditions .

What is the recommended protocol for Western blotting using ARHGEF18 antibodies?

For optimal Western blot results:

• Sample preparation: Prepare lysates from approximately 1 million cells per sample

• Gel selection: Use 8% acrylamide gels for optimal resolution of the 120-130 kDa ARHGEF18 protein

• Transfer: Perform overnight transfer to PVDF membrane for complete transfer of high molecular weight proteins

• Blocking: Block membranes in 1% BSA solution

• Primary antibody: Incubate with polyclonal rabbit or goat anti-ARHGEF18 antibody at 1 μg/ml in 0.1% BSA in TBS-T

• Secondary antibody: Use 1:10,000 dilution of donkey anti-rabbit or bovine anti-goat IgG conjugated to HRP

• Detection: Utilize enhanced chemiluminescence for visualization

• Loading control: Confirm equal loading with Ponceau staining of the membrane

• Quantification: Perform densitometric analysis using software such as ImageJ

This protocol has been validated with multiple biological replicates and is effective for detecting ARHGEF18 in various cellular contexts .

How should I optimize antigen retrieval for immunohistochemistry with ARHGEF18 antibodies?

For effective IHC staining:

• Primary recommendation: Use TE buffer at pH 9.0 for antigen retrieval

• Alternative approach: Citrate buffer at pH 6.0 can be used if TE buffer yields suboptimal results

• Validation tissues: The antibody has been validated on mouse kidney tissue, human kidney tissue, and human prostate cancer tissue

• Controls: Include positive control tissues (e.g., mouse testis) where ARHGEF18 expression has been confirmed

• Optimization: Test multiple dilutions within the recommended range (1:50-1:500) to determine optimal signal-to-background ratio for your specific tissue samples

Careful optimization of antigen retrieval conditions is particularly important for formalin-fixed, paraffin-embedded samples where protein cross-linking can mask epitopes .

How can I troubleshoot non-specific bands in Western blots using ARHGEF18 antibodies?

Non-specific bands may occur due to:

• Novel isoforms: Recent research has identified previously uncharacterized isoforms of ARHGEF18 (termed "LOCGEFs") that may appear as unexpected bands. The 1361-residue X3 isoform (NCBI XP_006722769.1) is most abundant in some cell types like eosinophils, while X4 and X5 isoforms may also be present .

• Cross-reactivity: If experiencing cross-reactivity, consider:

  • Increasing blocking time or concentration (try 5% BSA or milk)

  • Using more stringent washing conditions

  • Titrating antibody concentration to determine optimal dilution

  • Using alternative antibodies targeting different epitopes for validation

• Sample preparation issues:

  • Ensure complete protein denaturation

  • Add protease inhibitors to prevent degradation products

  • Optimize lysis buffer composition based on subcellular localization

• Cell/tissue-specific expression patterns:

  • Verify with literature whether your cell/tissue type expresses specific isoforms

  • Include appropriate positive controls (e.g., mouse testis tissue for WB)

How can I validate the specificity of ARHGEF18 antibodies for my research application?

Comprehensive validation approaches include:

• Multiple antibody comparison:

  • Use antibodies from different vendors targeting distinct epitopes

  • Compare polyclonal and monoclonal antibodies when available

  • Validate with both N-terminal and C-terminal targeting antibodies

• Genetic approaches:

  • Perform siRNA knockdown or CRISPR knockout of ARHGEF18

  • Overexpress tagged ARHGEF18 constructs for co-localization studies

• Recombinant protein controls:

  • Include purified recombinant ARHGEF18 as a positive control

  • Perform peptide competition assays with the immunogen peptide

• Cross-species validation:

  • Test reactivity in multiple species if studying conserved functions

  • Note that most antibodies show reactivity with human and mouse samples

• Application-specific validation:

  • For co-immunoprecipitation experiments, validate lack of interaction with similar GEFs

  • For GTPase pull-down assays, confirm specificity for RhoA vs. Rac1

What controls should I include when studying ARHGEF18 in different cell types?

For robust experimental design:

• Positive tissue/cell controls:

  • Mouse testis tissue for Western blot

  • Mouse kidney tissue, human kidney tissue, or human prostate cancer tissue for IHC

  • HEK-293 cells for immunofluorescence/ICC

• Negative controls:

  • Secondary antibody-only controls to assess background

  • Isotype controls to identify non-specific binding

  • Cells/tissues with confirmed low ARHGEF18 expression

• Cell-specific considerations:

  • For eosinophils, be aware that "LOCGEF" isoforms may be the predominant form, not the p114 isoform commonly studied in other cell types

  • For endothelial cells, consider controls exposed to different shear stress conditions, as ARHGEF18 activity varies with physiological versus pathological shear stress

• Functionality controls:

  • RhoA activation assays can serve as functional readouts for ARHGEF18 activity

  • Cytoskeletal organization assessments provide functional validation

How can I investigate ARHGEF18 specificity for different GTPases in my cell system?

To determine ARHGEF18's GTPase specificity:

• GTPase pull-down assays:

  • Use dominant-negative GTPase mutants (e.g., RhoA G17A, Rac1 G15A) that have high affinity for active GEFs

  • Perform pull-downs under different cellular conditions

  • Analyze co-precipitated proteins by immunoblotting for ARHGEF18

  • Include known GEF controls (e.g., Vav for Rac1) to validate assay performance

• Fluorescence-based GEF activity assays:

  • Measure nucleotide exchange rates on purified GTPases

  • Compare ARHGEF18 activity toward different GTPases (RhoA, Rac1, Cdc42)

• FRET-based biosensors:

  • Employ biosensors for different GTPases to measure activation in response to ARHGEF18 manipulation

  • Analyze spatial and temporal dynamics of activation

Data from epithelial cells and endothelial cells suggest ARHGEF18 preferentially activates RhoA rather than Rac1. In endothelial cells specifically, ARHGEF18 shows differential activity under varying shear stress conditions, with higher exchange activity for RhoA under physiological shear stress compared to pathological conditions .

How can I study the differential expression of ARHGEF18 isoforms across cell types?

To investigate isoform-specific expression:

• RNA analysis approaches:

  • Design primers spanning unique exon junctions of different isoforms

  • Perform RT-PCR to detect transcript presence

  • Use quantitative RT-PCR for relative abundance determination

  • Consider RNA-seq for comprehensive isoform profiling

• Protein-level investigations:

  • Select antibodies recognizing different epitopes specific to certain isoforms

  • For eosinophils, target the unique N-terminal regions of LOCGEF isoforms

  • Use immunoprecipitation followed by mass spectrometry for unbiased isoform identification

• Transcriptional regulation analysis:

  • Investigate cell-specific promoter usage using 5' RACE

  • Analyze chromatin accessibility at alternative promoters

  • Consider that LOCGEF isoforms may be controlled by leukocyte-specific transcription factors acting upstream of the canonical p114 isoform start site

Recent research has demonstrated that LOCGEFs (particularly the X3, X4, and X5 isoforms) are the predominant forms in human eosinophils, suggesting cell type-specific expression patterns that may have functional significance in specialized cellular contexts .

What approaches can I use to investigate ARHGEF18's role in RhoA-ROCK2 signaling pathways?

For comprehensive pathway analysis:

• Pharmacological approaches:

  • Use ROCK inhibitors (Y-27632, Fasudil) to probe downstream effects

  • Apply RhoA activators or inhibitors to establish pathway dependency

  • Employ actomyosin contractility inhibitors to assess cytoskeletal outcomes

• Genetic manipulation strategies:

  • Perform ARHGEF18 knockdown/knockout and assess RhoA activation status

  • Rescue experiments with wild-type vs. catalytically inactive ARHGEF18

  • Introduce constitutively active or dominant-negative RhoA to determine epistasis

• Functional readouts:

  • Measure stress fiber formation and actomyosin contractility

  • Assess cell-cell junction maturation in endothelial or epithelial cells

  • Examine eosinophil morphological changes (ovoid to acorn-shaped) following cytokine stimulation

  • Quantify integrin-mediated adherence to extracellular matrix proteins

• Spatiotemporal dynamics:

  • Use live-cell imaging with fluorescently tagged components

  • Analyze subcellular localization during activation events

  • Employ optogenetic approaches for precise pathway manipulation

ARHGEF18 has been implicated in maintaining neuro-epithelial apico-basal polarity, regulating cell-cell junction maturation, organizing actomyosin cytoskeletal components, and responding to mechanical forces in endothelial cells through the RhoA-ROCK2 signaling axis .

How can I investigate ARHGEF18's role in endothelial cell mechano-sensitivity?

To explore mechanotransduction roles:

• Shear stress experimental design:

  • Apply different shear stress conditions (physiological vs. pathological)

  • Measure ARHGEF18 activity using RhoA G17A pull-down assays

  • Compare responses between physiological shear stress (PSS) and pathological conditions (low shear stress [LSS] or high shear stress [HSS])

• ARHGEF18 localization studies:

  • Analyze subcellular redistribution in response to mechanical stimuli

  • Examine co-localization with mechanosensitive complexes

  • Investigate interactions with cell-cell junction proteins

• Downstream effector analysis:

  • Assess cytoskeletal remodeling responses

  • Measure barrier function under different mechanical conditions

  • Quantify RhoA activation spatiotemporally

• Molecular mechanism investigation:

  • Identify potential mechanosensitive domains through structure-function analysis

  • Investigate post-translational modifications occurring upon mechanical stimulation

  • Examine protein-protein interactions that may mediate mechanotransduction

Recent research indicates that ARHGEF18 exhibits differential exchange activity for RhoA under varying shear stress conditions, with higher activity observed under physiological shear stress compared to pathological conditions, suggesting a role in endothelial cell adaptation to mechanical forces .

What methodologies can I use to investigate the recently discovered "LOCGEF" isoforms of ARHGEF18?

To study novel LOCGEF isoforms:

• Isoform-specific detection:

  • Design primers targeting unique regions of X3 (XP_006722769.1), X4 (XP_011526140.1), and X5 (XP_011526141.1) isoforms

  • Develop antibodies against unique epitopes in these isoforms

  • Use mass spectrometry to identify peptides specific to different isoforms

• Functional characterization:

  • Express recombinant LOCGEFs in heterologous systems

  • Compare GEF activity of LOCGEFs versus canonical p114 isoform

  • Assess subcellular localization patterns specific to LOCGEF isoforms

• Regulatory mechanisms:

  • Investigate leukocyte-specific transcription factors controlling LOCGEF expression

  • Analyze promoter regions far upstream of the canonical p114 start site

  • Study differential regulation in response to inflammatory stimuli

• Cell-type specificities:

  • Compare expression across different leukocyte populations

  • Analyze functional relevance in eosinophil morphological changes and activation

  • Investigate potential roles in other immune cell types

Recent research has demonstrated that LOCGEFs are the predominant isoforms in human eosinophils, with the 1361-residue X3 isoform being most abundant. These findings challenge previous annotations in UniProtKB regarding ARHGEF18 Isoform 1 and suggest unique regulatory mechanisms for LOCGEF expression in leukocytes .

How can I correlate ARHGEF18 function with eosinophil morphological and functional changes?

To investigate this relationship:

• Cytokine stimulation experiments:

  • Treat eosinophils with IL-5 to induce rapid morphological changes (ovoid to acorn-shaped/polarized)

  • Monitor ARHGEF18 activity and localization during this 5-minute transformation

  • Assess changes in peptide abundance using isobaric labeling mass spectrometry

• Functional correlations:

  • Measure integrin-mediated adherence to extracellular matrix proteins

  • Analyze actomyosin cytoskeleton rearrangement

  • Assess ARHGEF18-RHOA-ROCK2-MLC signaling axis activation

• Genetic manipulation approaches:

  • Use siRNA to deplete specific LOCGEF isoforms

  • Perform rescue experiments with various ARHGEF18 constructs

  • Analyze resulting changes in morphology and function

• Spatiotemporal analysis:

  • Employ high-resolution microscopy to track changes in real-time

  • Co-visualize ARHGEF18 with cytoskeletal components during transformation

  • Correlate ARHGEF18 activity with specific stages of morphological change

Recent proteomics data indicated that peptides from LOC100996504 (now recognized as part of LOCGEF isoforms) decrease in abundance upon IL-5 activation of eosinophils, suggesting a dynamic role for these ARHGEF18 isoforms in eosinophil activation and function .