TBC1D24 Antibody

What is TBC1D24 and why is it important for neurological research?

TBC1D24 is a 559 amino acid cytoplasmic protein that functions as a GTPase-activating protein (GAP) for Rab family proteins . It contains a Rab-GAP TBC domain and a TLD domain that are essential for its interactions with other proteins, particularly ARF6 . TBC1D24 is highly expressed in the brain and plays crucial roles in neuronal projection development through negative modulation of ARF6 function, making it essential for proper neuronal growth and connectivity .

Defects in TBC1D24 are linked to familial infantile myoclonic epilepsy (FIME), a form of idiopathic epilepsy manifesting in early infancy with symptoms including myoclonic seizures, febrile convulsions, and tonic-clonic seizures . Additionally, recent research demonstrates that TBC1D24 regulates intraorganellar pH by positively modulating v-ATPase activity in neurons, suggesting that alterations in pH homeostasis could underlie TBC1D24-associated disorders . These critical neurological functions make TBC1D24 an important target for research on both neurological disorders and cellular signaling pathways.

What types of TBC1D24 antibodies are available for research applications?

Multiple types of TBC1D24 antibodies are available for research applications, primarily categorized by host species and antibody type:

• Mouse monoclonal antibodies: Such as the D-5 clone (IgM) from Santa Cruz Biotechnology that detects TBC1D24 in mouse, rat, and human samples .

• Rabbit polyclonal antibodies: Available from multiple vendors including Proteintech (25254-1-AP) and Abcam (ab272681, ab234723) .

These antibodies are developed using different immunogens - some target specific regions like the C-terminus (ab272681 targets aa 450 to C-terminus) or N-terminus (ab234723 targets aa 1-200), while others are raised against fusion proteins . This diversity provides researchers with options to select antibodies targeting different epitopes of TBC1D24 depending on experimental requirements and accessibility of epitopes in different applications.

TBC1D24 antibodies have been validated with various sample types:

• Cell lines: HEK-293, HeLa, RT4 (human urinary bladder cancer cell line), and BJ (human skin fibroblast) cells have been used successfully in western blot and immunofluorescence applications .

• Tissue samples: Human tissues including stomach, kidney, and pancreas have been validated for immunohistochemistry applications . TBC1D24 is highly expressed in the brain but also found in other tissues such as testis, skeletal muscle, heart, lung, and liver .

• Species reactivity: Most antibodies show reactivity with human samples, while some (like the Santa Cruz D-5 antibody) also detect mouse and rat TBC1D24 . The Proteintech antibody (25254-1-AP) has been cited in publications using both human and mouse samples .

When working with new sample types, optimization of protocols is recommended to ensure specific detection of TBC1D24.

How should researchers optimize western blot protocols for TBC1D24 detection?

Optimizing western blot protocols for TBC1D24 requires careful consideration of several parameters:

• Expected molecular weight: TBC1D24 has a calculated molecular weight of 63 kDa, but typically appears at 60-63 kDa on western blots . This slight variation may reflect post-translational modifications or isoform expression.

• Antibody selection and dilution: For the Proteintech antibody (25254-1-AP), a dilution range of 1:500-1:1000 is recommended . Sample-dependent optimization may be necessary for optimal results.

• Sample preparation: TBC1D24 is a cytoplasmic protein that interacts with membrane proteins like ARF6 and v-ATPase components . Proper cell lysis conditions ensuring extraction of membrane-associated proteins may improve detection.

• Loading controls: When studying TBC1D24 in brain tissues or neuronal cells, appropriate loading controls should be selected based on the subcellular fraction being analyzed, particularly when examining TBC1D24's interactions with membrane components like v-ATPase .

• Specificity verification: Using neutralizing peptides (such as sc-390377 P) can help confirm antibody specificity . Additionally, recombinant expression systems or knockout/knockdown models provide valuable controls for validating signal specificity.

For optimal results, researchers should follow antibody-specific protocols such as those provided by manufacturers. For example, Proteintech offers a specific western blot protocol for their TBC1D24 antibody (25254-1-AP) .

What are the considerations for immunofluorescence experiments with TBC1D24 antibodies?

When performing immunofluorescence experiments with TBC1D24 antibodies, researchers should consider:

• Fixation and permeabilization: PFA fixation with Triton X-100 permeabilization has been validated for TBC1D24 detection, as demonstrated with ab272681 in BJ cells . This approach maintains cellular architecture while allowing antibody access to the cytoplasmic protein.

• Antibody dilution: Different antibodies require specific dilutions for optimal results. For example, ab234723 has been validated at 1/100 dilution in HeLa cells, while ab272681 is effective at 2 μg/ml in BJ cells .

• Subcellular localization: TBC1D24 is primarily cytoplasmic but may associate with vesicular structures due to its role in vesicle trafficking and interaction with ARF6 . When studying TBC1D24's interaction with v-ATPase, co-localization experiments may reveal association with specific organelles .

• Controls: Including appropriate negative controls (secondary antibody only, isotype controls) and positive controls (cells known to express TBC1D24) is essential for validating staining patterns.

• Secondary antibody selection: Compatible secondary antibodies such as Alexa-Fluor®488-conjugated Goat Anti-Rabbit IgG (H+L) have been validated for rabbit polyclonal antibodies like ab234723 . For mouse monoclonal IgM antibodies like sc-390377, specific anti-mouse IgM secondaries are required .

• Counterstaining: Nuclear counterstains can help establish cellular architecture and provide context for TBC1D24 localization patterns.

How can researchers study TBC1D24's interaction with v-ATPase and its role in pH regulation?

Recent research has established TBC1D24's interaction with v-ATPase components and its role in regulating intraorganellar pH . To study these interactions:

• Co-immunoprecipitation assays: TBC1D24 has been shown to preferentially co-immunoprecipitate with kidney-specific ATP6V1B1 subunit and may also interact with the brain-enriched ATP6V1B2 subunit . Pull-down experiments in expression systems like COS-7 cells can be used with tagged versions of TBC1D24 (e.g., 3xFLAG-tagged TBC1D24) to investigate these interactions.

• pH-sensitive probes: To assess TBC1D24's functional impact on v-ATPase activity and pH regulation, researchers can employ pH-sensitive fluorescent probes targeted to specific organelles.

• Loss-of-function approaches: Given that TBC1D24 positively regulates v-ATPase activity in neurons, knockdown or knockout models can help elucidate how loss of TBC1D24 affects intracellular pH homeostasis .

• Rescue experiments: Testing whether wild-type TBC1D24 can rescue pH regulation defects in TBC1D24-deficient models, while disease-associated mutants cannot, may provide insight into pathological mechanisms.

• Trafficking assays: Since both TBC1D24 and v-ATPase are involved in vesicle trafficking, assays monitoring vesicle movement and fusion events can help clarify their functional relationship.

When designing these experiments, researchers should consider that brain tissue expresses the ATP6V1B2 subunit highly, while the ATP6V1B1 subunit is more kidney-specific . This tissue specificity may influence experimental outcomes and interpretation.

What are common causes of non-specific binding with TBC1D24 antibodies and how can they be addressed?

Non-specific binding with TBC1D24 antibodies can occur for various reasons:

• Cross-reactivity with related proteins: TBC1D24 contains a TBC domain shared with other TBC family proteins, which may lead to cross-reactivity. To address this:

  • Use antibodies validated for specificity, such as those tested against knockout samples

  • Include controls like neutralizing peptides (e.g., sc-390377 P)

  • Consider western blots to verify single-band specificity at the expected molecular weight (60-63 kDa)

• Inadequate blocking: Improper blocking can lead to high background. Optimize by:

  • Using BSA-containing buffers where appropriate (some antibodies like ab272681 are provided in solutions containing 0.1% BSA)

  • Extending blocking time or increasing blocking agent concentration

  • Testing different blocking agents (BSA, non-fat milk, serum)

• Suboptimal antibody dilution: Using too concentrated antibody can increase non-specific binding. For each antibody:

  • Follow manufacturer recommendations (e.g., 1:500-1:1000 for 25254-1-AP in WB)

  • Perform dilution series to determine optimal concentration for your specific samples

  • Be aware that optimal dilutions may differ between applications (WB vs. IHC)

• Inadequate washing: Insufficient washing can leave unbound antibody. Improve by:

  • Increasing wash duration and/or number of wash steps

  • Ensuring wash buffer completely covers samples

  • Adding low concentrations of detergent to wash buffers if appropriate

• Tissue-specific factors: Since TBC1D24 is expressed in multiple tissues, tissue-specific components may affect binding. Consider:

  • Optimizing antigen retrieval methods for IHC (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0 for 25254-1-AP)

  • Adjusting extraction conditions for different tissue types

How can researchers effectively validate TBC1D24 knockdown or knockout models using antibodies?

Validating TBC1D24 knockdown or knockout models using antibodies requires careful experimental design:

• Selection of appropriate antibodies:

  • Use antibodies that have been validated in western blot applications

  • Consider using multiple antibodies targeting different epitopes to confirm results

  • Antibodies like 25254-1-AP (targeting fusion protein) or ab272681 (targeting C-terminus) provide options for validation from different angles

• Controls and experimental design:

  • Include wild-type samples processed identically to knockout/knockdown samples

  • For siRNA or shRNA experiments, include scrambled/non-targeting controls

  • Consider using graduated knockdown approaches (dose-dependent siRNA) to show correlation between knockdown efficiency and phenotype

• Quantification approaches:

  • Normalize TBC1D24 signal to appropriate loading controls

  • Use digital image analysis software for unbiased quantification

  • Present data showing statistical significance of knockdown/knockout efficiency

• Functional validation:

  • Complement protein detection with functional assays related to TBC1D24's roles:

    • Neuronal projection development assays

    • ARF6 activity measurements

    • Vesicle trafficking assays

    • v-ATPase activity and pH regulation assessments

• Rescue experiments:

  • Re-express TBC1D24 in knockout models to confirm phenotype reversibility

  • Consider expressing disease-associated mutants to assess their functional impact

This comprehensive validation approach ensures that observed phenotypes are truly attributable to TBC1D24 deficiency rather than off-target effects or experimental artifacts.

What are the optimal approaches for studying TBC1D24's role in neuronal projection development?

TBC1D24 plays crucial roles in neuronal projection development, likely through negative modulation of ARF6 function . To study this function:

• Neuronal culture systems:

  • Primary neuron cultures (cortical, hippocampal) provide physiologically relevant models

  • Neuron-like cell lines (SH-SY5Y, PC12) can be used for initial screening

  • iPSC-derived neurons from patients with TBC1D24 mutations offer disease-relevant models

• Visualization techniques:

  • Immunofluorescence using validated antibodies like sc-390377 or ab234723 at optimized dilutions

  • Live imaging with fluorescently tagged TBC1D24 to track dynamics during neurite extension

  • Co-localization studies with markers for ARF6, actin, and vesicular components

• Quantitative analysis:

  • Neurite length, branching complexity, and growth cone morphology measurements

  • Time-lapse analysis of neurite extension rates and dynamics

  • Sholl analysis for dendritic arborization assessment

• Molecular manipulation approaches:

  • Knockdown/knockout of TBC1D24 using RNAi or CRISPR-Cas9

  • Expression of dominant-negative or constitutively active TBC1D24 mutants

  • Structure-function analysis by expressing domain-specific mutants (TBC domain vs. TLD domain)

• ARF6-focused experiments:

  • Measure ARF6 activation state (ARF6-GTP levels) in the presence/absence of TBC1D24

  • Express constitutively active or dominant negative ARF6 to test epistatic relationship with TBC1D24

  • Co-immunoprecipitation to confirm TBC1D24-ARF6 interaction in neuronal contexts

• Functional outcomes:

  • Assess synaptic formation and activity in mature neurons with manipulated TBC1D24 levels

  • Evaluate calcium signaling in developing neurites

  • Test whether v-ATPase activity and pH regulation contribute to TBC1D24's effects on neurite development

How can TBC1D24 antibodies be used to investigate its role in epilepsy and other neurological disorders?

TBC1D24 mutations are linked to familial infantile myoclonic epilepsy (FIME) and potentially other neurological disorders . Antibodies can facilitate research in this area through:

• Expression pattern analysis:

  • Compare TBC1D24 expression levels and localization in control versus epileptic brain tissues using immunohistochemistry with antibodies like ab234723 at 1/100 dilution

  • Assess changes in expression during development to understand critical periods for TBC1D24 function

  • Compare expression patterns across brain regions to identify particularly vulnerable circuits

• Patient-derived samples:

  • Analyze TBC1D24 protein levels in accessible patient samples (e.g., fibroblasts, lymphoblasts)

  • Use immunofluorescence to study subcellular localization changes in patient-derived cells

  • Examine post-translational modifications that might be altered in disease states

• Animal models:

  • Validate animal models of TBC1D24-related disorders using antibodies to confirm knockdown/knockout

  • Map TBC1D24 expression in brain sections from models with epilepsy-like phenotypes

  • Correlate TBC1D24 levels with seizure susceptibility or neuronal excitability

• Mechanistic investigations:

  • Study TBC1D24's interaction with ARF6 and v-ATPase in epilepsy models

  • Investigate whether pH dysregulation contributes to hyperexcitability phenotypes

  • Assess changes in synaptic vesicle trafficking that might contribute to epileptogenesis

• Potential therapeutic approaches:

  • Screen compounds that might stabilize mutant TBC1D24 or enhance residual function

  • Use antibodies to assess target engagement of therapeutic candidates

  • Monitor TBC1D24 expression changes in response to anti-epileptic treatments

When designing these studies, researchers should consider that TBC1D24's role may differ between acute seizure events and chronic epileptogenesis, necessitating temporal analysis of its function and regulation.

What are the most promising directions for studying TBC1D24's molecular interactions and signaling pathways?

TBC1D24 participates in multiple molecular interactions and signaling pathways that warrant further investigation:

• Rab protein regulation:

  • As a potential GTPase-activating protein for Rab family proteins, identify specific Rab targets using in vitro GTPase assays

  • Map interaction domains between TBC1D24 and Rab proteins

  • Study how these interactions affect vesicle trafficking in neurons and other cell types

• ARF6 modulation:

  • Further characterize how TBC1D24 negatively modulates ARF6 function

  • Investigate whether this modulation is direct or involves intermediate proteins

  • Determine how this interaction regulates actin dynamics during neuronal development

• v-ATPase complex interaction:

  • Expand upon the finding that TBC1D24 interacts with v-ATPase and regulates intraorganellar pH

  • Map the interacting domains between TBC1D24 and v-ATPase subunits

  • Investigate tissue-specific interactions (ATP6V1B1 in kidney vs. ATP6V1B2 in brain)

  • Study how disease-associated mutations affect this interaction

• Novel interaction partners:

  • Perform immunoprecipitation with TBC1D24 antibodies followed by mass spectrometry to identify new binding partners

  • Validate these interactions using complementary approaches (yeast two-hybrid, FRET, etc.)

  • Investigate how TBC1D24's TBC and TLD domains mediate different protein interactions

• Post-translational modifications:

  • Study how phosphorylation, ubiquitination, or other modifications regulate TBC1D24 function

  • Identify enzymes responsible for these modifications

  • Determine whether disease states alter TBC1D24's modification patterns

• Isoform-specific functions:

  • TBC1D24 exists in at least two alternatively spliced isoforms

  • Develop isoform-specific antibodies or detection strategies

  • Investigate whether different isoforms have distinct functions or tissue distributions

These research directions may yield valuable insights into TBC1D24's physiological roles and pathological mechanisms in disease states.

How can multiplexed approaches enhance TBC1D24 research in complex neuronal systems?

Complex neuronal systems require sophisticated approaches to dissect TBC1D24's functions:

• Multi-antibody immunofluorescence:

  • Combine TBC1D24 antibodies with markers for specific neuronal populations or subcellular compartments

  • Use antibodies like ab234723 (targeting N-terminal region) or ab272681 (targeting C-terminal region) with different fluorophore-conjugated secondaries

  • Include markers for ARF6, Rab proteins, v-ATPase components, and vesicular structures to study co-localization

• Proximity labeling approaches:

  • Express TBC1D24 fused to proximity labeling enzymes (BioID, APEX) to identify proteins in its vicinity

  • Compare proximity interactomes in different neuronal compartments or activity states

  • Validate interactions using co-immunoprecipitation with TBC1D24 antibodies

• Super-resolution microscopy:

  • Apply STED, STORM, or PALM imaging with TBC1D24 antibodies to visualize nanoscale localization

  • Study dynamic changes in localization during neuronal development or activity

  • Assess co-localization with interacting partners at nanoscale resolution

• Live imaging combined with fixed-cell analysis:

  • Use live imaging with fluorescently tagged TBC1D24 to track dynamics

  • Fix cells at critical timepoints for immunostaining with antibodies against endogenous proteins

  • Correlate live dynamics with molecular interactions at specific timepoints

• Single-cell approaches:

  • Combine immunofluorescence with single-cell transcriptomics to correlate TBC1D24 protein levels with gene expression profiles

  • Assess cell-to-cell variability in TBC1D24 expression and localization within neuronal populations

  • Identify cellular subpopulations particularly dependent on TBC1D24 function

• In vivo imaging approaches:

  • Use TBC1D24 antibodies for immunohistochemistry in brain sections from animal models

  • Correlate TBC1D24 expression with circuit-level functions or disease phenotypes

  • Develop in vivo labeling approaches for longitudinal studies

These multiplexed approaches can help unravel TBC1D24's complex functions in the context of neuronal circuits and networks, providing insights beyond what can be achieved with single-antibody approaches.

What quality control measures should researchers implement when working with TBC1D24 antibodies?

Implementing rigorous quality control measures ensures reliable results with TBC1D24 antibodies:

• Antibody validation:

  • Verify antibody specificity using positive controls (cells/tissues known to express TBC1D24) and negative controls (knockout/knockdown samples)

  • Use neutralizing peptides (like sc-390377 P) to confirm specificity

  • Consider multiple antibodies targeting different epitopes to confirm findings

• Lot-to-lot consistency:

  • Test new antibody lots against previous lots to ensure consistent performance

  • Maintain reference samples for comparative analysis

  • Document lot numbers in research notes and publications

• Application-specific controls:

  • For Western blotting: Include molecular weight markers and verify the detected band matches the expected size (60-63 kDa)

  • For immunofluorescence: Include secondary-only controls and isotype controls

  • For immunohistochemistry: Include negative control tissues and peptide competition controls

• Sample preparation quality:

  • Ensure consistent sample preparation to minimize variability

  • Validate protein extraction efficiency, especially when comparing different tissue types

  • Monitor sample degradation that might affect epitope integrity

• Documentation and reporting:

  • Record detailed antibody information (manufacturer, catalog number, lot number, dilution)

  • Document all experimental conditions thoroughly

  • Include representative images of controls in publications

  • Report both positive and negative results to build a comprehensive understanding of antibody performance

By implementing these measures, researchers can increase confidence in their results and contribute to reproducible TBC1D24 research across the scientific community.

How should researchers integrate antibody-based approaches with other methodologies to comprehensively study TBC1D24?

A comprehensive understanding of TBC1D24 requires integration of multiple methodologies:

• Combining protein and gene expression analysis:

  • Correlate protein levels detected with antibodies to mRNA expression

  • Investigate potential post-transcriptional regulation mechanisms

  • Assess whether mutations affect protein stability versus expression

• Structural insights and antibody epitope mapping:

  • Use structural biology approaches to understand TBC1D24's domains

  • Map epitopes recognized by different antibodies

  • Interpret antibody-based results in the context of protein structure

• Functional assays:

  • Complement localization studies with functional readouts:

    • GTPase activity assays for Rab protein regulation

    • ARF6 activation assays

    • v-ATPase activity and pH measurements

    • Neurite outgrowth and morphology analysis

• Systems biology approaches:

  • Place TBC1D24 in the context of broader protein networks

  • Use antibodies for immunoprecipitation followed by mass spectrometry

  • Correlate TBC1D24 levels with global proteomic or transcriptomic changes

• Genetic approaches:

  • CRISPR-Cas9 editing to create cellular models with TBC1D24 mutations

  • Rescue experiments with wild-type and mutant constructs

  • Patient-derived cells harboring natural TBC1D24 variants

• In vivo significance:

  • Translate in vitro findings to animal models

  • Use antibodies to validate model systems

  • Correlate molecular findings with behavioral or electrophysiological outcomes

This integrated approach provides a more complete picture of TBC1D24's functions and disease relevance than any single methodology alone, helping to translate molecular insights into potential therapeutic strategies for TBC1D24-related disorders.

What are the emerging areas of research where TBC1D24 antibodies may have significant impact?

Several emerging research areas may benefit significantly from TBC1D24 antibodies:

• Neurotherapeutics for TBC1D24-related disorders:

  • Screening compounds that stabilize mutant TBC1D24 protein

  • Monitoring TBC1D24 levels during therapeutic interventions

  • Developing biomarkers for patient stratification and treatment response

• pH dysregulation in neurological diseases:

  • Further investigating TBC1D24's role in regulating v-ATPase and intraorganellar pH

  • Examining whether pH dysregulation contributes to epileptogenesis or neurodegeneration

  • Developing pH-targeted interventions for TBC1D24-deficient conditions

• Non-neuronal functions of TBC1D24:

  • Exploring TBC1D24's roles in testis, skeletal muscle, heart, kidney, lung, and liver

  • Investigating tissue-specific interaction partners and functions

  • Addressing potential systemic manifestations of TBC1D24 mutations

• Developmental neurobiology:

  • Temporal analysis of TBC1D24 expression during brain development

  • Role in neuronal migration, axon guidance, and synaptogenesis

  • Contribution to circuit formation and refinement

• Precision medicine approaches:

  • Correlating specific TBC1D24 mutations with protein expression, localization, and function

  • Developing mutation-specific therapeutic strategies

  • Using antibodies to monitor treatment efficacy in personalized approaches

• Extracellular vesicle research:

  • Given TBC1D24's roles in vesicle trafficking, investigating its potential presence in exosomes

  • Exploring whether TBC1D24 or its fragments could serve as biomarkers in biofluids

  • Examining intercellular communication mediated by TBC1D24-containing vesicles