ARL2 Antibody Pair

What is ARL2 and why is it an important research target?

ARL2 (ADP-Ribosylation Factor-Like Protein 2) is a small GTP-binding protein that cycles between inactive GDP-bound and active GTP-bound forms, regulated by guanine nucleotide exchange factors (GEF) and GTPase-activating proteins (GAP) . It serves as a critical research target due to its multifunctional roles in cellular processes including:

• Regulation of microtubule dynamics and centrosome integrity

• Mitochondrial fusion and function

• Nuclear processes including homologous recombination repair

• Cortical neuronal development

Methodological approach: When initiating ARL2 research, begin with subcellular fractionation followed by Western blotting to identify its distribution patterns, as ARL2 is found in both cytosolic and mitochondrial compartments , with recent evidence also pointing to nuclear localization .

What specific applications are ARL2 antibodies validated for?

ARL2 antibodies have been validated for multiple experimental applications depending on the specific antibody:

• Western Blotting (WB)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunocytochemistry/Immunofluorescence (ICC/IF)

• Flow Cytometry (intracellular)

How should researchers distinguish between ARL2 and other ARL family proteins?

To ensure specificity when studying ARL2:

• Select antibodies validated against related proteins - high-quality ARL2 antibodies should be tested for cross-reactivity with human ARF1-6, ARL1, and ARL3

• Include appropriate controls:

  • Positive control: Purified recombinant ARL2 protein

  • Negative control: Cell lysates with ARL2 knockdown

• Target unique epitopes - the C-terminal region of ARL2 (sequence LDDISSRIFTAD) provides higher specificity compared to more conserved domains

• Validate findings with multiple antibodies targeting different epitopes

What methodological considerations are important when studying ARL2's mitochondrial functions?

For investigating ARL2's role in mitochondria:

• Subcellular fractionation protocol:

  • Use protease digestion to distinguish outer membrane-associated from internal mitochondrial proteins

  • ARL2 is found in a protease-resistant form within mitochondria

  • Validate fractionation quality with markers (e.g., mtHSP70, cytochrome c)

• Functional analysis workflow:

  • ARL2 siRNA knockdown leads to mitochondrial fragmentation, perinuclear clustering, and ~50% loss of cellular ATP

  • Use dominant negative ARL2[T30N] mutant to separate morphological from ATP production effects

  • Pair with ELMOD2 knockdown studies to differentiate between ARL2's dual mitochondrial pathways

• Protein interaction studies:

  • ARL2·GTP·BART complex specifically binds to ANT1 but not ANT2 isoforms

  • Validate using tissues from ANT1-knockout models as controls

  • Use overlay assays for detecting binding partners following the methods described by Clark et al.

How can ARL2 antibodies be utilized to investigate homologous recombination repair mechanisms?

Based on recent discoveries about ARL2's nuclear functions in DNA repair:

• Expression correlation analysis:

  • ARL2 expression positively correlates with all six RAD51 family genes essential for homologous recombination repair (HRR)

• Cancer stem cell (CSC) investigation protocol:

  • ARL2 is induced in colon cancer stem cells

  • Differential requirements: ARL2 depletion causes M phase arrest in non-CSCs but DNA break stress accumulation and apoptosis in CSCs

• Chromatin-associated function detection:

  • Develop chromatin fractionation protocols to isolate ARL2 in nuclear compartments

  • Use immunofluorescence with DNA damage markers to visualize ARL2 recruitment to damage sites

  • Employ co-immunoprecipitation with RAD51 family proteins to elucidate interaction mechanisms

What are the optimal protocols for detecting ARL2 in neuronal tissue samples?

For neuronal studies investigating ARL2's role in cortical development:

• In vivo analysis:

  • In utero electroporation (IUE) followed by immunohistochemistry reveals ARL2's function in neuronal migration

  • ARL2 knockdown results in significant cell retention in the ventricular zone (VZ), subventricular zone (SVZ), and intermediate zone (IZ)

• Subcellular distribution quantification:

  • Develop zone-specific quantification protocols (similar to those reporting 30.94% VZ, 27.64% SVZ, 38.00% IZ, and 3.42% CP localization after knockdown)

• Microtubule dynamics assessment:

  • ARL2 is critical for microtubule growth in mouse neural progenitor cells (mNPCs)

  • Loss of ARL2 causes a shift from symmetric to asymmetric division patterns

What are the critical controls for ARL2 antibody-based experiments?

Implement these essential controls for rigorous ARL2 research:

• Antibody validation controls:

  • Peptide competition assay using the immunizing peptide (e.g., C-terminal peptide LDDISSRIFTAD)

  • Knockdown/knockout lysates for specificity confirmation

  • Cross-reactivity testing against related proteins

• Functional analysis controls:

  • For GTP-dependence studies: Compare ARL2·GTP versus ARL2·GDP binding

  • For mitochondrial studies: Include both ARL2 and ELMOD2 knockdowns to differentiate pathways

  • For homologous recombination: Compare with RAD51 family knockdowns

• Tissue-specific comparisons:

  • ARL2 expression varies across tissues, with highest expression in brain and colon cancer tissues

  • Include multiple tissue types when analyzing antibody specificity in tissue samples

How can researchers optimize ELISA protocols using ARL2 antibody pairs?

For developing sensitive and specific sandwich ELISA:

• Optimized sandwich ELISA protocol:

  • Capture antibody: Unconjugated rabbit polyclonal anti-ARL2

  • Detection antibody: Biotin-conjugated rabbit polyclonal anti-ARL2

  • Buffer conditions: 0.01 M PBS, pH 7.4

• Critical parameters:

  • Antibody dilution optimization through checkerboard titration

  • Extended incubation at 4°C to improve sensitivity for low-abundance samples

  • Background reduction using specialized blocking buffers containing 50% glycerol

• Validation strategy:

  • Use recombinant ARL2 for standard curve generation

  • Cross-validate with Western blot quantification

  • Control for potential interference from ARL2-binding proteins (BART, tubulin, ANT)

What methodological approaches can address contradictory findings in ARL2 research?

To resolve conflicting observations about ARL2 functions:

• Compartment-specific analysis:

  • ARL2 functions differently in cytosol, mitochondria, and nucleus

  • Design experiments that specifically isolate effects in each compartment

  • Use targeted constructs with compartment-specific localization signals

• Binding partner dependency:

  • ARL2's functions depend on specific binding partners in different contexts

  • BART·ARL2·GTP complex binds ANT1 but not ANT2

  • Systematically analyze binding partner expression in your experimental system

• Cell-type specific requirements:

  • ARL2 depletion affects cancer stem cells differently than non-stem cancer cells

  • ARL2 requirements vary between neural progenitors and differentiated neurons

  • Include multiple cell types when studying ARL2 functions to account for context-dependent effects

How can researchers investigate ARL2's role in neural development using antibody-based approaches?

To explore ARL2's neuronal functions:

• Developmental timeline analysis:

  • Track ARL2 expression during cortical development using immunohistochemistry

  • Correlate with neural progenitor cell division patterns

  • Study impact on symmetric vs. asymmetric divisions in neural stem cells

• Migration assay protocol:

  • In utero electroporation followed by immunohistochemistry reveals ARL2's function in neuronal migration

  • Quantify distribution across ventricular zone, subventricular zone, intermediate zone, and cortical plate

  • Use co-staining with cell cycle markers to correlate with proliferation status

• Cytoskeletal interaction studies:

  • Analyze ARL2-microtubule interactions in developing neurons

  • Investigate impact on neuronal polarization and axon formation

  • Combine with tubulin markers for co-localization studies

What is the recommended methodology for studying ARL2 in cancer research applications?

For cancer-focused ARL2 investigations:

• Expression analysis approach:

  • ARL2 is highly expressed in breast and colon cancer compared to other cancers

  • mRNA expression is significantly higher in colon cancer tissues than adjacent normal tissues

  • Combine tissue microarray analysis with public database mining (TCGA, Human Protein Atlas)

• Cancer stem cell protocol:

  • Isolate cancer stem cell populations and compare ARL2 levels with non-stem cancer cells

  • ARL2 is induced in colon cancer stem cells and required for their maintenance

  • Study relationship with RAD51 family genes essential for homologous recombination repair

• Nuclear function investigation:

  • In cancer tissues, ARL2 is predominantly localized in the nucleoplasm

  • Develop chromatin fractionation protocols to isolate nuclear ARL2

  • Analyze correlation between ARL2 levels and DNA damage response in cancer samples

How should researchers approach the study of ARL2 in relation to therapeutic applications?

For translational research focusing on ARL2:

• Therapeutic target validation:

  • ARL2 and homologous recombination repair pathways represent potential therapeutic targets specific for colon cancer stem cells

  • Use colony formation and sphere-forming assays to assess cancer stem cell-specific vulnerabilities

• Combination strategy assessment:

  • Test ARL2 inhibition alongside DNA-damaging agents

  • Evaluate synthetic lethality with PARP inhibitors based on ARL2's role in homologous recombination

  • Analyze differential responses in cancer stem cells versus bulk tumor cells

• Biomarker development:

  • Validate ARL2 as a potential prognostic marker in colon cancer

  • Correlate expression levels with clinical outcomes

  • Develop immunohistochemistry protocols with appropriate cutoff values for clinical applications

How can ARL2 antibody techniques be integrated with other molecular approaches?

For comprehensive ARL2 function analysis:

• Integrated workflow:

  • Combine antibody-based detection with genetic manipulation (CRISPR/siRNA)

  • Correlate protein levels with transcriptomic changes after ARL2 modulation

  • Map protein interactions using immunoprecipitation followed by mass spectrometry

• Structure-function correlation:

  • Use antibodies recognizing specific conformational states (GTP vs. GDP-bound)

  • Combine with site-directed mutagenesis of key ARL2 residues

  • Develop in vitro binding assays to quantify interaction with partners like BART and ANT1

• Dynamic analysis protocols:

  • Live-cell imaging with fluorescently tagged ARL2 for studying subcellular trafficking

  • Photoactivation studies to track movement between cellular compartments

  • FRAP (Fluorescence Recovery After Photobleaching) to analyze binding dynamics