RHOT2 Antibody

What is RHOT2 and why are RHOT2 antibodies important in mitochondrial research?

RHOT2 (Ras homolog gene family member T2), also known as MIRO-2 or hMiro-2, belongs to the mitochondrial Rho GTPase family. It is a crucial protein localized to the outer membrane of mitochondria and plays key roles in mitochondrial dynamics and transport. RHOT2 antibodies are essential tools for investigating mitochondrial function and dynamics due to their ability to specifically detect this protein in various experimental contexts. Current research indicates RHOT2 is involved in calcium-dependent regulation of mitochondrial movement and may have implications in neurodegenerative conditions such as Parkinson's disease . When designing experiments to study mitochondrial transport, RHOT2 antibodies enable visualization of this critical protein through multiple detection methods including Western blotting, immunofluorescence, and immunohistochemistry .

What are the key differences between monoclonal and polyclonal RHOT2 antibodies in experimental applications?

Monoclonal and polyclonal RHOT2 antibodies differ significantly in their experimental utility and application specificity:

Monoclonal RHOT2 antibodies (like 68469-1-Ig):

• Recognize a single epitope on the RHOT2 protein, providing high specificity

• Demonstrate consistent lot-to-lot reproducibility, critical for longitudinal studies

• Typically show narrower reactivity across species (often human-specific)

• Optimal for applications requiring precise epitope targeting, as in the case of 68469-1-Ig which is validated primarily for Western blot applications with dilution ranges of 1:5000-1:50000

Polyclonal RHOT2 antibodies (like 11237-1-AP):

• Recognize multiple epitopes on the RHOT2 protein, offering higher sensitivity

• Show broader cross-reactivity across species (human, mouse, rat)

• Particularly effective for applications requiring signal amplification

• Can be used across multiple applications including WB (1:1000-1:10000), IHC (1:50-1:500), IF/ICC (1:50-1:500), and IP (0.5-4.0 μg)

When designing experiments requiring detection of potentially low abundance RHOT2 in tissue samples, polyclonal antibodies often provide better sensitivity, while monoclonal antibodies offer advantages in experiments requiring consistent epitope recognition over time.

How should storage and handling conditions be optimized for RHOT2 antibodies to maintain detection sensitivity?

Proper storage and handling of RHOT2 antibodies is critical for maintaining their specificity and sensitivity in experimental applications. Based on manufacturer recommendations across multiple products:

Storage conditions:

• Store RHOT2 antibodies at -20°C for long-term preservation

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

• Antibody 60464-3-PBS requires storage at -80°C due to its PBS-only buffer formulation

• Avoid repeated freeze-thaw cycles which can lead to antibody degradation and loss of activity

Buffer formulations:

• Most RHOT2 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• This buffer composition helps maintain stability during freeze-thaw cycles

• For antibodies requiring conjugation (like 60464-3-PBS), azide-free and BSA-free formulations are available

For experimental methodologies requiring maximum antibody performance, consider the following research-validated approaches:

• Aliquot antibodies upon receipt to minimize freeze-thaw cycles

• Use sterile technique when handling to prevent microbial contamination

• Centrifuge antibody vials briefly before opening to collect solution at the bottom of the vial

• When working with diluted antibody solutions, prepare them fresh for each experiment

These handling procedures are essential for maintaining detection sensitivity, particularly in experiments targeting endogenous RHOT2 expression levels in primary cells or tissue samples.

What are the optimal sample preparation protocols for detecting RHOT2 in different cellular compartments?

Since RHOT2 is a mitochondrial outer membrane protein, sample preparation must preserve its native localization while allowing antibody accessibility. The following methodological approaches have been validated in research settings:

For Western blot applications:

• Cell lysis should be performed using mild detergents (like 1% Triton X-100) that preserve membrane protein integrity

• Include protease inhibitors in lysis buffers to prevent degradation

• Enrichment of mitochondrial fractions may enhance detection sensitivity

• The observed molecular weight of RHOT2 ranges from 68-80 kDa depending on post-translational modifications

For immunofluorescence/immunocytochemistry:

• Mild fixation (4% paraformaldehyde for 10-15 minutes) preserves RHOT2 epitopes

• Brief permeabilization (0.1-0.2% Triton X-100 for 5-10 minutes) allows antibody access without extracting membrane proteins

• Co-staining with established mitochondrial markers (e.g., TOM20, MitoTracker) confirms specificity of RHOT2 localization

• RHOT2 antibodies like 11237-1-AP have been validated for IF/ICC at dilutions of 1:50-1:500

For immunohistochemistry on fixed tissue:

• Antigen retrieval is critical; both citrate buffer (pH 6.0) and TE buffer (pH 9.0) methods have been validated

• For 11237-1-AP and NBP2-93326 antibodies, high-pressure antigen retrieval with 10mM citrate buffer (pH 6.0) is recommended

• Optimal dilutions range from 1:50-1:500 depending on the specific antibody and tissue type

When performing co-localization studies, these methodological considerations are essential for accurate determination of RHOT2 interactions with other mitochondrial or cytoskeletal proteins.

How can phosphosite-specific antibodies be utilized to investigate RHOT2 regulation mechanisms?

Phosphosite-specific antibodies represent an advanced approach for studying RHOT2 regulation through post-translational modifications. Building on methodologies developed for other membrane proteins like dopamine receptors , researchers can employ similar techniques for RHOT2:

Methodological approach:

• Identification of putative phosphorylation sites through phosphoproteomic analysis or prediction algorithms

• Generation of phospho-specific antibodies using double phosphorylated peptides corresponding to specific regions of RHOT2

• Validation using phosphatase treatments to confirm specificity for phosphorylated versus non-phosphorylated forms

• Application in Western blotting to monitor changes in phosphorylation state under different cellular conditions

For RHOT2 regulation studies, researchers can adapt the approach used for D2R phosphorylation detection:

• Use λ-phosphatase treatment as a negative control to confirm phospho-specificity

• Compare basal versus stimulated phosphorylation states

• Employ kinase inhibitors to identify responsible kinases

A sample experimental design based on D2R phosphorylation study methodologies :

This methodological framework enables researchers to investigate how RHOT2 phosphorylation states affect mitochondrial dynamics and transport in response to calcium signaling and other cellular pathways.

What strategies can resolve conflicting RHOT2 antibody reactivity between different detection systems?

When researchers encounter conflicting results between different detection methods using RHOT2 antibodies, systematic troubleshooting and validation approaches are essential:

• Epitope accessibility differences:

  • Western blot detects denatured proteins while IHC/IF detect proteins in their native conformation

  • Use antibodies targeting different epitopes for cross-validation

  • The RHOT2 antibody 11237-1-AP (targeting fusion protein Ag1752) may detect different epitopes than 68469-1-Ig (targeting fusion protein Ag29690)

• Validation through genetic approaches:

  • Include RHOT2 knockout or knockdown controls in experiments

  • 11237-1-AP has been validated in knockout/knockdown applications as indicated in published literature

  • Compare results with overexpression systems using tagged RHOT2 constructs

• Cross-platform validation methodology:

  Detection Method	Sample Preparation	Epitope Concern	Validation Approach
  Western Blot	Denatured protein	Linear epitopes	RHOT2-KO control lysate
  Immunofluorescence	Fixed cells/tissues	Conformational	Co-localization with mitochondrial markers
  Immunohistochemistry	Fixed, embedded tissue	Masked epitopes	Antigen retrieval optimization
  Immunoprecipitation	Native protein	Accessible surface	Pre-clearing optimization

• Application-specific considerations:

  • For Western blot: The observed molecular weight may vary (68-80 kDa) based on post-translational modifications

  • For IHC: Antigen retrieval methods significantly impact epitope accessibility; both citrate buffer (pH 6.0) and TE buffer (pH 9.0) have been validated

  • For IP applications: Pre-clearing lysates and optimizing antibody concentrations (0.5-4.0 μg for 1.0-3.0 mg of protein) are critical

By implementing these systematic validation strategies, researchers can resolve discrepancies and ensure reliable RHOT2 detection across experimental platforms.

How can RHOT2 antibodies be utilized to investigate mitochondrial dynamics in Parkinson's disease models?

RHOT2 has emerged as a potential factor in Parkinson's disease (PD) pathogenesis through its role in mitochondrial transport and quality control. Researchers can employ RHOT2 antibodies in PD research through the following methodological approaches:

• Genetic association studies have investigated RHOT2 variation in PD risk:

  • The search results indicate that RHOT2 genetic variation has been analyzed in relation to PD risk, though cumulative effects were inconsistent across different statistical tests (N variants = 40; CMC p = 0.044, Zeggini p = 0.166, MB p = 0.404, SKAT p = 0.125, SKAT-O p = 0.210, Fp p = 0.659)

  • This genetic foundation provides rationale for protein-level investigations

• Experimental design for RHOT2 protein analysis in PD models:

  • Compare RHOT2 expression levels between control and PD patient-derived samples using validated antibodies

  • Analyze RHOT2 localization in relation to damaged mitochondria in neurotoxin-based or genetic PD models

  • Investigate RHOT2 interactions with PINK1/Parkin pathway components using co-immunoprecipitation with antibodies validated for this application like 11237-1-AP

• Tissue-specific analysis strategy:

  • The RHOT2 antibody 11237-1-AP has been validated for detection in multiple human tissues including colon, kidney, heart, and lung cancer tissues

  • RHOT2 antibody NBP2-93326 has been validated for IHC in rat kidney and mouse brain tissues, making it suitable for neurological research

  • These validations enable comparative studies between brain regions affected in PD versus unaffected regions

• Cell-type specific analysis:

  • RHOT2 antibodies have been validated in multiple cell lines including neuronal models

  • Use immunofluorescence to compare RHOT2 distribution in dopaminergic versus non-dopaminergic neurons

  • Combine with markers for mitochondrial fragmentation to assess correlation with disease pathology

This methodological framework enables researchers to investigate whether RHOT2 dysfunction contributes to the mitochondrial transport defects observed in PD pathogenesis, potentially revealing new therapeutic targets.

What controls should be implemented when studying RHOT2 protein interactions in disease contexts?

When investigating RHOT2 protein interactions in disease contexts, rigorous controls are essential for ensuring experimental validity and reproducibility:

• Antibody specificity controls:

  • Include RHOT2 knockout or knockdown samples as negative controls

  • For polyclonal antibodies like 11237-1-AP, which has been validated in KD/KO applications, this control is particularly informative

  • Pre-absorption controls using the immunizing peptide can validate antibody specificity

• Co-immunoprecipitation controls:

  • The 11237-1-AP antibody has been validated for co-immunoprecipitation (CoIP) applications

  • Include IgG-matched isotype controls for non-specific binding assessment

  • Reverse Co-IP (using antibodies against the interacting partner to pull down RHOT2)

  • Input controls (5-10% of lysate used for IP) to verify protein expression

• Disease-specific experimental design:

  Disease Context	Positive Control	Negative Control	Validation Method
  Parkinson's Disease	PINK1/Parkin interactions	RHOT2 knockdown	Reciprocal Co-IP
  Mitochondrial dysfunction	Known RHOT2 interactors (e.g., TRAK1/2)	Competition with excess peptide	Proximity ligation assay
  Neurodegeneration	Age-matched healthy tissue	Non-neuronal tissue	Mass spectrometry validation

• Methodological controls for phosphorylation studies:

  • Include phosphatase treatment controls as demonstrated in the D2R phosphorylation study

  • Use both phospho-specific and total RHOT2 antibodies for normalization

  • Include kinase inhibitor treatments to confirm pathway specificity

• Cell type and subcellular localization controls:

  • Include mitochondrial markers (TOM20, MitoTracker) to confirm RHOT2 localization

  • Use mitochondrial fractionation to enrich for RHOT2 and its interacting partners

  • Compare disease-relevant cell types with non-relevant cells as biological controls

By implementing these systematic controls, researchers can ensure that observed RHOT2 interactions are specific, reproducible, and relevant to the disease context under investigation.

How can researchers optimize Western blot protocols for detecting endogenous versus overexpressed RHOT2?

Detecting endogenous versus overexpressed RHOT2 requires different optimization strategies due to expression level differences. The following methodological guidelines address common challenges:

For endogenous RHOT2 detection:

• Antibody selection and dilution:

  • Use high-sensitivity antibodies like 11237-1-AP (1:1000-1:10000) or 68469-1-Ig (1:5000-1:50000)

  • Begin with the more concentrated end of the dilution range for endogenous detection

  • Polyclonal antibodies may offer greater sensitivity for low abundance proteins

• Sample preparation:

  • Enrich for mitochondrial fractions to concentrate RHOT2 content

  • Load higher protein amounts (40-80 μg) for tissue samples

  • Include phosphatase inhibitors to preserve post-translational modifications

• Detection system:

  • Use high-sensitivity ECL substrates or fluorescent secondary antibodies

  • Consider longer exposure times while monitoring background

  • The observed molecular weight varies between 68-80 kDa depending on the antibody used

For overexpressed RHOT2 detection:

• Antibody dilution:

  • Use more diluted antibody concentrations (upper end of recommended range)

  • For 68469-1-Ig, dilutions up to 1:50000 may be appropriate

  • Consider using antibodies against expression tags if available

• Sample preparation:

  • Load less protein (10-20 μg) to prevent signal saturation

  • Include untransfected controls for comparison

  • Short exposure times to prevent oversaturation

• Special considerations:

  • Monitor for potential aggregation or degradation of overexpressed protein

  • Verify subcellular localization matches endogenous pattern through fractionation

Common troubleshooting approach:

These methodological approaches ensure accurate detection of RHOT2 regardless of expression level context.

What are the critical parameters for successful immunofluorescence detection of RHOT2 in primary neurons?

Detecting RHOT2 in primary neurons requires careful optimization due to the complex morphology and sensitivity of neuronal cells. Based on validated antibody protocols and neuronal imaging best practices:

• Fixation and permeabilization parameters:

  • Mild fixation: 4% paraformaldehyde for 10-15 minutes at room temperature

  • Gentle permeabilization: 0.1% Triton X-100 for 5-10 minutes to preserve mitochondrial membrane integrity

  • Alternative: 100% ice-cold methanol for 10 minutes for simultaneous fixation and permeabilization

• Antibody selection and optimization:

  • The 11237-1-AP antibody has been validated for IF/ICC applications at dilutions of 1:50-1:500

  • NBP2-93326 is recommended at 1:50-1:200 dilution for IF/ICC applications

  • Extended primary antibody incubation (overnight at 4°C) improves signal-to-noise ratio

• Neuronal-specific considerations:

  • Block with 5-10% normal serum plus 1% BSA to reduce non-specific binding

  • Include cytoskeletal markers (MAP2, Tau) to visualize neuronal processes

  • Co-stain with mitochondrial markers (TOM20, MitoTracker) to confirm RHOT2 localization

  • Image both cell bodies and distal neurites to assess mitochondrial distribution

• Advanced imaging parameters:

  • Perform z-stack acquisition (0.3-0.5 μm steps) to capture three-dimensional distribution

  • Use deconvolution algorithms to improve resolution of individual mitochondria

  • Consider super-resolution techniques for detailed mitochondrial morphology analysis

• Critical controls:

  • Include RHOT2 siRNA knockdown neurons as negative controls

  • Compare with fibroblasts where RHOT2 detection has been well-established

  • Process control samples with primary antibody omission

Methodological validation table for neuronal RHOT2 imaging:

By carefully optimizing these parameters, researchers can achieve specific and sensitive detection of RHOT2 in primary neurons, enabling detailed analysis of mitochondrial dynamics in neuronal compartments.

Available RHOT2 Antibodies and Their Validated Applications