RAB35 Antibody

Which tissue or cell types show high expression of RAB35?

RAB35 is ubiquitously expressed, but particularly high levels have been detected in brain tissue. Western blot analyses have confirmed strong RAB35 expression in human brain tissue, mouse brain tissue, and rat brain tissue . Among cell lines, positive detection has been reported in A431 cells, A375 cells, HeLa cells, MCF-7 cells, RAW 264.7 cells, U-87 MG cells, and neuroblastoma cell lines including SK-N-SH, IMR32, and SK-N-AS .

What applications are RAB35 antibodies suitable for?

RAB35 antibodies have been validated for multiple applications including:

How can I optimize antigen retrieval for RAB35 immunohistochemistry?

For optimal antigen retrieval in paraffin-embedded tissues, there are two recommended methods:

• TE buffer at pH 9.0 (preferred method)

• Citrate buffer at pH 6.0 (alternative method)

The choice between these methods may depend on your specific tissue type and fixation protocol. For formalin-fixed tissues, the higher pH TE buffer often provides better epitope unmasking for RAB35 detection. If using paraformaldehyde fixation, fresh preparation is critical as long-term stored PFA turns into formalin as the molecules congregate, which can affect antigen preservation and accessibility .

Why might I observe different subcellular localization patterns with RAB35 antibodies?

RAB35 exhibits dynamic localization patterns depending on its activation state and the cellular context. It can be found on:

• The plasma membrane

• Intracellular vesicles (particularly those positive for transferrin receptor)

• The immunological synapse (in activated T cells)

If observing inconsistent localization patterns, consider:

• The GTP/GDP-bound state of RAB35 (active vs. inactive)

• Cell type-specific differences in RAB35 effectors

• Fixation methods that may differentially preserve membrane structures

• Antibody epitope accessibility in different subcellular compartments

For immunofluorescence studies particularly in T cells, note that Rab35 strongly localizes to the immunological synapse upon antigen recognition, with temporal concordance to TCR-ζ recruitment .

How do I troubleshoot non-specific bands when using RAB35 antibodies?

When encountering non-specific bands in Western blot applications:

• Validation strategy: Use RAB35 knockout samples as negative controls. Published data shows complete absence of the 23-25 kDa band in RAB35 knockout HeLa cells .

• Blocking optimization: For polyclonal antibodies, 5% BSA in TBS-T has been reported to provide cleaner results than milk-based blocking solutions .

• Antibody specificity verification:

  • Different epitope-targeting antibodies may show varying specificity

  • N-terminal vs. middle region vs. C-terminal antibodies can have different non-specific binding profiles

• Sample preparation: Additional centrifugation steps to remove membrane fragments can reduce non-specific membrane protein interactions .

What is the role of RAB35 in receptor recycling and how can antibodies help investigate this function?

RAB35 functions as an essential rate-limiting regulator of the fast recycling pathway back to the plasma membrane. Specific experimental approaches to investigate this role include:

• Co-localization studies: RAB35 antibodies can be used in conjunction with transferrin receptor antibodies to assess co-localization in recycling endosomes. Published data shows substantial co-localization between RAB35 and transferrin receptor in intracellular vesicles .

• Functional assays: Combining RAB35 immunofluorescence with transferrin uptake and export assays can reveal the functional consequences of RAB35 manipulation. EPI64C (a RAB35 GAP) and RAB35 dominant negative constructs have been shown to impair transferrin export from recycling pathways in T-cells .

• Vacuolar phenotype assessment: Disruption of RAB35 function (through dominant negative constructs or GAP overexpression) induces large vacuoles marked by transferrin receptor, which can be visualized using appropriate antibodies .

How does RAB35 contribute to immunological synapse formation and T cell function?

RAB35 plays a critical role in immunological synapse (IS) formation between T cells and antigen-presenting cells (APCs). Research approaches using RAB35 antibodies to study this include:

• Dynamic recruitment analysis: Time-lapse imaging studies have demonstrated that RAB35 is strongly recruited to the IS upon antigen recognition, with temporal concordance to TCR-ζ recruitment .

• Conjugate formation assessment: Flow cytometry and microscopy approaches have shown that disruption of RAB35 function (through dominant negative constructs or EPI64C expression) significantly impairs T cell-APC conjugate formation .

• TCR enrichment quantification: Surface TCR enrichment at the IS can be quantified by comparing TCR intensity at the synapse versus the opposite pole of the cell. RAB35 dominant negative constructs significantly impair this enrichment .

• Mechanistic studies: RAB35 may regulate synapse formation by controlling polarized secretion from recycling endosomes, which can be investigated using co-localization studies with SNARE proteins involved in TCR-polarized secretion .

What controls should I include when validating a new RAB35 antibody?

A comprehensive validation strategy for RAB35 antibodies should include:

For advanced validation, consider using multiple RAB35 antibodies targeting different epitopes and confirming consistent results across detection methods.

How can I design experiments to study RAB35 activation state using antibodies?

While standard RAB35 antibodies detect total RAB35 regardless of activation state, several approaches can be used to study the GTP-bound (active) versus GDP-bound (inactive) states:

• Co-immunoprecipitation with effector proteins: Active RAB35-GTP preferentially binds to its effectors, allowing for isolation of the active pool .

• Combination with dominant negative (S22N) and constitutively active (Q67L) mutants: These can serve as controls for inactive and active states, respectively .

• GAP protein interactions: EPI64C (TBC1D10C) is a Rab35-specific GAP that can be used in combination with RAB35 antibodies to modulate and study activation states .

• In vitro GAP assays: Using recombinant RAB35 and GAP proteins with [γ-32P]GTP to measure GTPase activity, followed by Western blot confirmation with RAB35 antibodies .

What methodological considerations are important when using RAB35 antibodies for live-cell imaging?

When conducting live-cell imaging studies of RAB35:

• The native RAB35 antibody is not suitable for live-cell applications as it cannot penetrate the intact plasma membrane.

• Alternative approaches include:

  • Using fluorescently tagged RAB35 constructs (RAB35-GFP or RAB35-YFP) for transfection-based studies

  • For endogenous protein tracking, consider cell-permeable nanobodies if available

  • Fixed timepoint studies using RAB35 antibodies after fixation at defined timepoints

• For T cell immunological synapse studies, live-cell imaging has revealed that Rab35 and TCR-ζ are highly colocalized at the synapse, with temporal concordance in their recruitment .

How do RAB35 antibodies help investigate its role in neurological processes and disease models?

RAB35 is highly expressed in neuronal tissues and has been implicated in neurite outgrowth regulation. Research approaches include:

• Expression profiling: RAB35 antibodies have confirmed high expression in human, mouse, and rat brain tissues .

• Neuronal subcellular localization: Immunofluorescence studies in neuroblastoma cell lines (SK-N-SH, IMR32, SK-N-AS) and primary neurons can reveal compartment-specific localization .

• Neurodegenerative disease models: RAB35 antibodies have been used in studies of retinal ganglion cell response to optic nerve crush, a model relevant to glaucoma research .

• Neuronal differentiation: The role of RAB35 in neurite outgrowth can be assessed by combining morphological analyses with RAB35 immunostaining in differentiating neuronal cells .

What approaches are used to investigate RAB35 in glucose transport and insulin signaling?

RAB35 has been implicated in the regulation of insulin-induced glucose transporter SLC2A4/GLUT4 translocation to the plasma membrane in adipocytes. Research strategies include:

• Co-localization studies: RAB35 antibodies can be used alongside GLUT4 antibodies to assess potential co-trafficking in response to insulin stimulation .

• TBC1D13 interactions: This protein works together with RAB35 in regulating GLUT4 translocation, and their interaction can be studied through co-immunoprecipitation approaches using RAB35 antibodies .

• Translocation assays: The effect of RAB35 manipulation (knockdown, overexpression, or mutant expression) on insulin-stimulated GLUT4 translocation can be quantified using surface biotinylation and RAB35 antibody-based detection methods.