RAB33A Antibody

What is RAB33A and what are its primary cellular functions?

RAB33A is a member of the RAS oncogene family, belonging to the small GTPase superfamily and Rab family. It has a calculated molecular weight of approximately 27 kDa and is also known as RABS10 or Small GTP-binding protein S10 . RAB33A functions primarily in vesicular transport mechanisms, particularly in neuronal cells. In cultured rat hippocampal neurons, RAB33A mediates anterograde vesicular transport along growing axons .

Research has demonstrated that RAB33A is localized to the Golgi apparatus and to synaptophysin-positive vesicles that are transported along axons . Functionally, RAB33A plays critical roles in:

• Mediating anterograde transport of post-Golgi synaptophysin-positive vesicles

• Contributing to membrane insertion at growth cones and axon outgrowth

• Interacting with singar1/RUFY3, which suppresses formation of surplus axons in neurons

• Inducing non-canonical autophagy in certain cancer cells

Which tissues and cell types express RAB33A?

RAB33A expression demonstrates tissue specificity that researchers should consider when planning experiments:

This expression pattern indicates that RAB33A has tissue-specific functions and may play different roles in normal physiology versus pathological conditions.

What applications are RAB33A antibodies validated for?

Based on the available data, RAB33A antibodies have been validated for several experimental applications:

When selecting a RAB33A antibody for your research, consider the specific application, species reactivity, and validated dilution ranges to ensure optimal experimental outcomes.

How does RAB33A contribute to cancer metastasis, and how can antibodies help investigate this mechanism?

Recent research has uncovered a critical role for RAB33A in promoting cancer metastasis, particularly in cervical cancer. Mechanistically, RAB33A promotes metastasis through:

• Enhancing RhoC accumulation through a non-canonical autophagy mechanism

• Stabilizing RhoC by preventing its degradation

• Facilitating pseudopodia formation, which contributes to cancer cell invasion

For researchers investigating this pathway, RAB33A antibodies can be applied in multiple experimental approaches:

When studying these pathways, researchers should consider using multiple antibodies targeting different epitopes of RAB33A to validate findings.

What are the best experimental designs for studying RAB33A's role in neuronal development?

When investigating RAB33A's function in neuronal development, consider these methodological approaches:

• Live-cell imaging with fluorescently tagged RAB33A:

  • Transfect neurons with EGFP-RAB33A constructs to monitor vesicle movement in real-time

  • Use confocal microscopy with appropriate environmental controls (37°C)

  • Combine with synaptophysin markers to study vesicular co-transport

• RNAi knockdown experiments:

  • Use vector-based RNAi with validated targeting sequences (e.g., 5'-CAGGACAAGAACGCTTCCGTA-3' corresponding to nucleotides 278-298 in rat RAB33A)

  • Include appropriate negative controls

  • Analyze phenotypes such as axon length, growth cone dynamics, and vesicular transport

• Co-localization studies:

  • Use RAB33A antibodies together with markers for the Golgi apparatus (giantin, GM130, adaptin γ, TGN38) and other Rab proteins

  • Apply Golgi-disrupting agents like Brefeldin A to confirm Golgi localization

  • Quantify co-localization using appropriate statistical measures

• Pseudocolor ratio imaging:

  • Combine RAB33A immunoreactivity with volume markers like CMFDA for standardized quantification

  • Analyze relative accumulation in growth cones and other neuronal compartments

These approaches will provide complementary data on RAB33A's role in neurodevelopment and vesicular transport.

How should researchers interpret conflicting RAB33A localization patterns in different experimental systems?

When facing contradictory RAB33A localization data, consider these methodological approaches:

• Cell type-specific differences:

  • RAB33A shows differential localization between neuronal and non-neuronal cells

  • In neurons, RAB33A localizes to both Golgi apparatus and synaptophysin-positive vesicles

  • In cancer cells, RAB33A may show more diverse subcellular distribution patterns

• Isoform specificity:

  • Ensure antibodies are specific to RAB33A rather than other family members like RAB33B

  • Verify epitope mapping and validate with positive and negative controls

  • Consider using multiple antibodies targeting different regions of RAB33A

• Activation state considerations:

  • Like other Rab GTPases, RAB33A cycles between GTP-bound (active) and GDP-bound (inactive) forms

  • These states may show different localization patterns

  • Consider using antibodies that recognize specific activation states

• Experimental validation approaches:

  • Perform subcellular fractionation to biochemically confirm localization

  • Use super-resolution microscopy to resolve ambiguous localization patterns

  • Validate with both overexpression and knockdown experiments

  • Include proper controls for antibody specificity using RAB33A knockout cells

When reporting results, clearly document the experimental conditions, cell types, and antibody clones used to facilitate interpretation across studies.

What are the optimal fixation and permeabilization conditions for RAB33A immunostaining?

For successful RAB33A immunofluorescence staining, consider these protocol recommendations:

For co-localization studies with Golgi markers, ensure that fixation conditions preserve Golgi structure. When performing double or triple immunostaining, carefully select antibodies raised in different host species to avoid cross-reactivity.

How should researchers troubleshoot non-specific binding in RAB33A Western blots?

When encountering non-specific bands in RAB33A Western blots, implement these systematic troubleshooting strategies:

• Antibody validation and controls:

  • Verify antibody specificity using RAB33A-overexpressing and knockout samples

  • Include positive control tissues known to express RAB33A (brain tissue works well)

  • Use recombinant RAB33A protein as a molecular weight reference

• Optimization of blocking conditions:

  • Test different blocking agents (5% non-fat milk, 3-5% BSA)

  • Extend blocking time to reduce background

  • Consider adding 0.1% Tween-20 to wash buffers

• Dilution optimization:

  • Test a range of antibody dilutions (1:2000-1:10000 for Western blotting)

  • Reduce primary antibody concentration if non-specific binding persists

  • Optimize secondary antibody dilutions

• Remember that RAB33A:

  • Has a calculated molecular weight of 27 kDa

  • May show slightly different migration patterns depending on post-translational modifications

  • Can be detected in various tissues, with strongest signal in brain samples

• Sample preparation considerations:

  • Ensure complete protein denaturation

  • Include protease inhibitors in lysis buffers

  • Consider phosphatase inhibitors if studying phosphorylation states

What experimental controls are essential when studying RAB33A-mediated autophagy?

When investigating RAB33A's role in autophagy, particularly non-canonical autophagy, include these critical controls:

• Autophagy flux controls:

  • Compare conditions with and without lysosomal inhibitors (Bafilomycin A1, Chloroquine)

  • RAB33A overexpression increases LC3-II levels, which is further enhanced by Baf A1 or CQ treatment

  • Monitor p62/SQSTM1 accumulation as an additional autophagy flux marker

• Pathway validation controls:

  • Include 3-MA (PI3K inhibitor) to distinguish RAB33A-induced non-canonical autophagy (3-MA resistant) from canonical autophagy

  • Test dependency on key autophagy regulators by knockdown/knockout of:

    • ATG5, ATG7, ATG16L1 (core autophagy machinery)

    • Beclin1, WIPI2 (dispensable for RAB33A-induced autophagy)

• Visualization controls:

  • Use GFP-RFP-LC3 tandem reporters to assess autophagosome-lysosome fusion

  • RAB33A overexpression increases the colocalization of GFP and RFP puncta, indicating accumulation of autophagosomes

  • Include electron microscopy to visualize autophagosome structures

• Molecular interaction controls:

  • Analyze RAB33A interaction with TBC1D2A, which it uses to inactivate RAB7

  • Include RAB7 activity assays to confirm mechanism

  • Use RAB33A mutants (constitutively active or dominant negative) to verify GTPase activity dependence

These controls will help distinguish RAB33A-mediated non-canonical autophagy from other autophagy pathways and confirm the specificity of observed effects.

How can RAB33A antibodies be used to investigate potential therapeutic targets in cancer?

RAB33A represents a promising therapeutic target, particularly in cancer metastasis. Researchers can use RAB33A antibodies to:

• Stratify patient populations:

  • Use IHC scoring with RAB33A antibodies to identify patients with high expression

  • These patients may benefit from targeted therapies, as high RAB33A expression correlates with poorer prognosis in cervical cancer

  • Develop standardized scoring systems based on staining intensity and percentage of positive cells

• Validate downstream targets:

  • RhoC stabilization is a key mechanism through which RAB33A promotes metastasis

  • Use co-immunoprecipitation with RAB33A antibodies to identify additional interacting partners

  • Investigate how RAB33A affects non-canonical autophagy to identify targetable nodes

• Monitor treatment response:

  • Develop assays to measure RAB33A activity or expression levels before and after treatment

  • Identify biomarkers that correlate with RAB33A-mediated processes

  • Use paired antibodies to RAB33A and its targets to assess pathway inhibition

• Explore combination therapies:

  • The findings suggest that "RhoC inhibitors may be beneficial for treating cervical cancer patients with high levels of RAB33A"

  • Investigate synergistic effects between RAB33A pathway inhibition and standard treatments

• Study resistance mechanisms:

  • Analyze changes in RAB33A expression or localization in treatment-resistant cells

  • Investigate compensatory pathways that may emerge when RAB33A is inhibited

This research direction could lead to the development of personalized medicine approaches for cancer patients with high RAB33A expression.

What techniques can be used to study the dynamic interaction between RAB33A and its binding partners?

To investigate the dynamic interactions between RAB33A and its binding partners like Singar1/RUFY3 and TBC1D2A, consider these advanced techniques:

• Proximity-based protein interaction assays:

  • FRET (Förster Resonance Energy Transfer) using fluorescently tagged RAB33A and binding partners

  • BiFC (Bimolecular Fluorescence Complementation) to visualize interactions in living cells

  • PLA (Proximity Ligation Assay) using antibodies against RAB33A and interaction partners

• Live cell imaging approaches:

  • Use fluorescently tagged proteins to track dynamic interactions in real-time

  • Apply photobleaching techniques (FRAP, FLIP) to assess mobility and exchange rates

  • Combine with pharmacological interventions to test dependency on specific pathways

• In vitro biochemical characterization:

  • Use purified recombinant proteins to measure binding kinetics via SPR or BLI

  • Conduct GTPase activity assays to determine how interactions affect RAB33A function

  • Map interaction domains through deletion and point mutants

• Mass spectrometry-based approaches:

  • Employ BioID or APEX proximity labeling to identify proteins in the vicinity of RAB33A

  • Use crosslinking mass spectrometry to capture transient interactions

  • Perform quantitative proteomics to map changes in the interactome under different conditions

• Super-resolution microscopy:

  • Apply techniques like STED, STORM, or PALM to visualize co-localization at nanoscale resolution

  • Combine with particle tracking to follow movement of vesicles containing RAB33A and partners

These approaches will provide complementary data on the spatial and temporal dynamics of RAB33A interactions and their functional significance.

How can researchers differentiate between the functions of RAB33A and its closely related family member RAB33B?

Distinguishing between RAB33A and RAB33B functions requires careful experimental design:

• Expression pattern analysis:

  • RAB33A is predominantly expressed in brain, lymphocytes, and melanocytes

  • RAB33B has a more ubiquitous expression pattern

  • Use tissue-specific contexts to isolate isoform-specific functions

• Antibody selection and validation:

  • Choose antibodies that specifically recognize RAB33A without cross-reactivity to RAB33B

  • Validate antibody specificity using overexpression and knockout controls

  • Consider generating isoform-specific antibodies if commercial options show cross-reactivity

• Genetic manipulation approaches:

  • Use siRNA or CRISPR targeting unique sequences in RAB33A and RAB33B

  • Perform rescue experiments with wildtype and mutant constructs

  • Create cell lines with individual or double knockouts to study compensatory mechanisms

• Functional assays:

  • RAB33A functions in axonal transport and cancer metastasis through non-canonical autophagy

  • RAB33B has established roles in canonical autophagy and retrograde Golgi transport

  • Design assays that specifically measure these distinct processes

• Structural biology approach:

  • Analyze differences in protein structure and binding interfaces

  • Identify isoform-specific binding partners through differential interactome analysis

  • Use structural information to develop isoform-selective inhibitors

By implementing these strategies, researchers can delineate the specific functions of RAB33A versus RAB33B and avoid confounding results due to overlapping functions or antibody cross-reactivity.

What are the optimal storage conditions for maintaining RAB33A antibody activity?

Proper storage is crucial for maintaining antibody activity and preventing degradation:

Additional storage recommendations:

• Aliquot antibodies upon receipt to minimize freeze-thaw cycles

• For antibodies without glycerol, add sterile glycerol to a final concentration of 30-50% before storage at -20°C

• Store in non-frost-free freezers to avoid temperature fluctuations

• Include date of first use and number of freeze-thaw cycles on each aliquot

Following these guidelines will help maintain antibody performance for reproducible results across experiments.

How should researchers select the appropriate RAB33A antibody for their specific application?

When selecting a RAB33A antibody, consider these parameters based on your experimental needs:

• Application compatibility:

  • For Western blot: Antibodies that recognize denatured epitopes (e.g., 68389-1-Ig, recommended dilution 1:2000-1:10000)

  • For IF/ICC: Antibodies validated for recognizing native conformation (e.g., 68389-1-Ig, recommended dilution 1:200-1:800)

  • For IHC: Antibodies validated for paraffin sections (e.g., GTX 55915, recommended dilution 1:100)

• Species reactivity:

  • Most commercial RAB33A antibodies react with human, mouse, and rat RAB33A

  • Some also cross-react with pig and rabbit samples

  • Verify cross-reactivity if working with less common model organisms

• Immunogen and epitope information:

  • For polyclonal antibodies, check immunogen sequence for potential cross-reactivity

  • For A12448, the immunogen corresponds to amino acids 173-218 of Human RAB33A

  • Consider epitope accessibility in native versus denatured protein

• Clonality considerations:

  • Monoclonal antibodies (e.g., 68389-1-Ig, clone 1C7F11) offer consistency between lots

  • Polyclonal antibodies may provide stronger signals through recognition of multiple epitopes

• Validation evidence:

  • Review validation data including Western blot images, IF/ICC images

  • Check publications citing the antibody

  • Consider antibodies validated using knockout controls

• Format needs:

  • Unconjugated format for most standard applications

  • Consider directly conjugated antibodies for multicolor flow cytometry or IF

By systematically evaluating these factors, researchers can select the most appropriate RAB33A antibody for their specific experimental needs.

How might RAB33A antibodies contribute to understanding neurodegenerative diseases?

Given RAB33A's role in neuronal development and vesicular transport, RAB33A antibodies can be valuable tools for investigating neurodegenerative disease mechanisms:

• Axonal transport defects:

  • RAB33A mediates anterograde transport of synaptophysin-positive vesicles along axons

  • Disrupted axonal transport is implicated in multiple neurodegenerative diseases

  • RAB33A antibodies can help visualize and quantify changes in vesicular transport dynamics

• Autophagy dysfunction:

  • RAB33A induces non-canonical autophagy

  • Autophagy defects are common in neurodegenerative diseases including Alzheimer's and Parkinson's

  • Investigating RAB33A's role in neuronal autophagy may reveal disease-relevant mechanisms

• Golgi fragmentation:

  • RAB33A localizes to the Golgi apparatus

  • Golgi fragmentation occurs in multiple neurodegenerative conditions

  • RAB33A antibodies can help characterize Golgi structural changes

• Protein aggregation studies:

  • Non-canonical autophagy induced by RAB33A may affect clearance of protein aggregates

  • Co-localization studies using RAB33A antibodies with disease-specific aggregates could reveal novel interactions

• Biomarker development:

  • Changes in RAB33A expression or localization might serve as disease biomarkers

  • Multi-parameter analysis including RAB33A could improve diagnostic precision

These applications could provide new insights into the pathogenesis of neurodegenerative diseases and potentially identify novel therapeutic targets.

What role might RAB33A play in immune cell function, and how can antibodies help elucidate these mechanisms?

RAB33A is expressed in lymphocytes , suggesting potential roles in immune function that warrant investigation:

• Vesicular transport in immune cells:

  • RAB proteins regulate vesicular trafficking essential for immune cell functions

  • RAB33A antibodies can help map vesicular compartments in various immune cell types

  • Co-localization with immune synapse markers could reveal roles in directed secretion

• Cytokine secretion pathways:

  • The Golgi apparatus, where RAB33A localizes , is critical for cytokine processing

  • Investigate whether RAB33A regulates secretory pathways in immune cells

  • Use RAB33A antibodies in combination with cytokine staining to visualize trafficking

• Autophagy in immune response:

  • RAB33A's role in non-canonical autophagy may affect immune cell functions

  • Autophagy regulates antigen presentation, inflammasome activation, and pathogen clearance

  • Study how RAB33A expression correlates with autophagy markers in immune contexts

• Lymphocyte activation and differentiation:

  • Monitor RAB33A expression and localization during lymphocyte activation

  • Compare RAB33A distribution in naïve versus memory T cells

  • Investigate potential correlations with activation markers

• Immune cell migration:

  • Given RAB33A's role in promoting cancer cell motility through RhoC stabilization

  • Examine whether similar mechanisms operate in immune cell migration

  • Correlate RAB33A expression with migration capacity in different immune cell subsets

These studies would expand our understanding of RAB33A beyond its established neuronal and cancer-related functions to potential roles in immune regulation.