RAB27A Antibody

What is RAB27A and why is it significant in cellular research?

RAB27A is a member of the small GTPase superfamily, specifically belonging to the Rab family. It functions as a membrane-bound protein involved in protein transport and small GTPase-mediated signal transduction pathways. The significance of RAB27A in cellular research stems from its critical role in regulating vesicle trafficking, particularly in the fusion of vesicles or organelles with target membranes . Specifically, RAB27A plays essential functions in cytotoxic granule exocytosis in lymphocytes and is required for both granule maturation and granule docking and priming at the immunologic synapse . Mutations in the RAB27A gene are associated with Griscelli syndrome type 2, a rare autosomal recessive disorder characterized by pigment dilution and uncontrolled T-lymphocyte and macrophage activation .

How can I confirm the specificity of a RAB27A antibody?

Confirming antibody specificity is crucial for reliable research outcomes. Several validation methods are recommended:

• Western blot analysis using knockdown cell lines: Compare parental cell lines with RAB27A knockdown lines to verify specificity. For example, U-87 MG human glioblastoma/astrocytoma parental cells should show a specific band at approximately 26 kDa that is significantly reduced in RAB27A knockdown U-87 MG cells .

• Cross-reactivity testing: Perform direct ELISAs with both RAB27A and related proteins like RAB27B. High-quality antibodies show minimal cross-reactivity (less than 1% with recombinant human RAB27B) .

• Immunocytochemistry with positive and negative control cell lines: Use cell lines known to express RAB27A (such as SK-Mel-28 human malignant melanoma cells) as positive controls and those that don't express RAB27A (like Daudi human Burkitt's lymphoma cells) as negative controls .

• Simple Western or traditional Western blot analysis with purified proteins or lysates from tissues known to express RAB27A, such as human prostate tissue or K562 human chronic myelogenous leukemia cell line .

What are the typical applications for RAB27A antibodies in research?

RAB27A antibodies can be employed in multiple experimental applications:

• Western Blot analysis: For detecting RAB27A protein expression levels in cell or tissue lysates. The protein typically appears at approximately 26-32 kDa depending on the experimental system .

• Immunocytochemistry/Immunofluorescence: For visualizing subcellular localization of RAB27A, typically showing cytoplasmic staining patterns .

• ELISA: For quantitative detection of RAB27A in solution .

• Simple Western analysis: For automated, size-based detection of RAB27A in complex samples .

• Knockout/knockdown validation studies: For confirming antibody specificity and studying RAB27A function .

How should I optimize RAB27A antibody dilutions for different applications?

Optimal antibody dilution varies by application and must be determined empirically. Based on validated protocols:

Begin with the manufacturer's recommended dilution, then perform a dilution series to determine optimal concentration for your specific sample type. Always include appropriate positive and negative controls to ensure specificity and minimize background staining .

What are the critical factors for successful RAB27A detection in melanoma cell lines?

Detection of RAB27A in melanoma cell lines requires special consideration due to its role in melanosomal transport:

• Sample preparation: Complete lysis of melanoma cells requires robust lysis buffers that can disrupt melanosome structures. Immunoblot Buffer Group 1 has been validated for RAB27A detection under reducing conditions .

• Selection of appropriate melanoma cell lines: SK-Mel-28 human malignant melanoma cell line has been confirmed to express high levels of RAB27A and serves as an excellent positive control .

• Subcellular localization: In melanoma cells, RAB27A localizes predominantly to the cytoplasm with a characteristic punctate staining pattern reflecting its association with melanosomes. NorthernLights™ 557-conjugated secondary antibodies provide good visualization when counterstained with DAPI .

• Controls: Include both melanoma cell lines (positive control) and non-melanocytic cell lines such as Daudi human Burkitt's lymphoma (negative control) to confirm staining specificity .

• Fixation methods: Immersion fixation techniques preserve RAB27A epitopes better than other fixation methods in melanoma cells .

How can I properly reconstitute and store RAB27A antibodies to maintain optimal activity?

Proper handling of RAB27A antibodies is essential for maintaining their functionality:

• Reconstitution protocols:

  • For lyophilized antibodies: Add the specified volume of sterile PBS (typically to achieve 0.2-1 mg/mL final concentration)

  • For small pack sizes: These may be supplied either lyophilized or as a 0.2 μm filtered solution in PBS

• Storage recommendations:

  • Store lyophilized antibodies at +4°C until reconstitution

  • After reconstitution, aliquot and store at -20°C to -80°C

  • Avoid repeated freeze-thaw cycles

  • Do not freeze lyophilized antibodies prior to reconstitution

• Stabilization considerations:

  • Some formulations include albumin as a stabilizer

  • Working dilutions should be prepared fresh before use

  • Buffer solutions typically consist of phosphate buffered saline with preservatives like 0.09% Sodium Azide

How can RAB27A antibodies be employed to investigate the role of RAB27A in colorectal cancer progression?

RAB27A has emerged as a potential biomarker and therapeutic target in colorectal cancer (CRC), though with some conflicting reports in the literature:

What are the optimal conditions for detecting RAB27A in exosome preparations?

Exosomes are extracellular vesicles involved in intercellular communication, and RAB27A plays a crucial role in their secretion:

• Exosome isolation protocol compatibility: Standard exosome isolation methods (ultracentrifugation, precipitation, or size exclusion) are compatible with subsequent RAB27A detection. Exosome Standards (K562) have been validated as positive controls for RAB27A detection .

• Sample preparation: Exosome lysates should be prepared using buffers that effectively solubilize membrane proteins. Load approximately 0.5 mg/mL of exosome lysate for optimal detection .

• Detection methods:

  • Simple Western: Has been validated for RAB27A detection in exosome preparations using the 12-230 kDa separation system, with RAB27A appearing at approximately 32 kDa

  • Western blot: Standard protocols can be used with appropriate positive controls

  • Immunogold electron microscopy: For visualizing RAB27A on individual exosomes

• Quantification considerations: When comparing RAB27A levels across different exosome preparations, normalization to exosome markers like CD63 or CD9 is essential for accurate interpretation of results.

How can I differentiate between RAB27A and RAB27B when investigating vesicle trafficking?

RAB27A and RAB27B are closely related isoforms with distinct but overlapping functions in vesicle trafficking:

• Antibody selection: Choose antibodies specifically validated for non-cross-reactivity between RAB27A and RAB27B. For example, the Synaptic Systems antibody (Cat. No. 168 013) is specifically affinity-purified using the non-conserved C-terminal part of RAB27A to ensure specificity, with documented lack of cross-reactivity to RAB27B .

• Epitope considerations: The recombinant immunogen used to generate the antibody should target non-conserved regions, such as AA 1 to 221 from rat RAB27A (UniProt Id: P23640) .

• Validation methods:

  • Direct ELISA comparing recombinant RAB27A and RAB27B should show <1% cross-reactivity

  • Western blot with recombinant proteins or tissues with differential expression

  • Knockout/knockdown validation: Some antibodies have been validated using RAB27A knockdown cell lines

• Compensatory expression: In some conditions (e.g., Griscelli syndrome caused by RAB27A mutations), RAB27B expression may be upregulated to compensate for loss of RAB27A function. This biological compensation must be considered when interpreting experimental results .

What are common issues in RAB27A Western blotting and how can they be resolved?

Western blotting for RAB27A may encounter several technical challenges:

• Inconsistent band sizes: RAB27A typically appears at 26-32 kDa depending on the experimental system. Variability may be due to:

  • Post-translational modifications

  • Different reducing conditions (use consistent reducing conditions; Immunoblot Buffer Group 1 has been validated)

  • Sample preparation methods

• Non-specific bands: To reduce background and increase specificity:

  • Optimize antibody dilution (typically 2-2.5 μg/mL for cell lysates)

  • Increase blocking time/concentration

  • Use RAB27A knockdown samples as negative controls to identify specific bands

  • Consider HRP-conjugated secondary antibodies for cleaner results

• Low signal intensity: To improve signal:

  • Use PVDF membrane rather than nitrocellulose for better protein retention

  • Ensure complete transfer (verify with Ponceau staining)

  • Increase protein loading (recommended range: 0.2-0.5 mg/mL)

  • Optimize exposure time

• Sample selection: Use validated positive controls such as human prostate tissue, SK-Mel-28 cells, or K562 cells known to express RAB27A .

How should I design experiments to investigate RAB27A's role in cytotoxic granule exocytosis in immune cells?

Investigating RAB27A's function in cytotoxic granule exocytosis requires careful experimental design:

• Cell model selection: Choose appropriate immune cell models:

  • Primary cytotoxic T lymphocytes (CTLs)

  • Natural killer (NK) cells

  • CD8+ T cell lines

• Functional assays:

  • Cytotoxicity assays (e.g., chromium release assay, flow cytometry-based killing assays)

  • Degranulation assays measuring surface expression of LAMP1/CD107a

  • Live-cell imaging of granule movement and fusion

• Immunofluorescence approach:

  • Co-staining with RAB27A antibody (3 μg/mL) and markers of cytotoxic granules (perforin, granzymes)

  • Use NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody or similar fluorescent conjugates

  • Counterstain with DAPI to visualize nuclei

  • Confocal microscopy to assess colocalization at the immunological synapse

• Genetic manipulation controls:

  • RAB27A knockdown/knockout cells

  • Rescue experiments with wild-type vs. mutant RAB27A

  • Griscelli syndrome patient-derived cells as natural RAB27A-deficient models

• Biochemical approaches:

  • Immunoprecipitation to identify RAB27A-interacting proteins in immune cells

  • GTP-binding assays to assess RAB27A activation state during immune cell activation

How can RAB27A antibodies be used to investigate the relationship between RAB27A and Griscelli syndrome?

Griscelli syndrome type 2 (GS2) is directly linked to mutations in the RAB27A gene. RAB27A antibodies can be valuable tools in GS2 research:

• Diagnostic applications:

  • Western blot analysis of patient-derived cells to detect altered RAB27A protein levels or sizes

  • Immunofluorescence to assess subcellular localization changes in melanocytes and immune cells

• Mutation characterization:

  • Different RAB27A mutations may result in truncated proteins, altered GTPase activity, or misfolding

  • Western blot with antibodies recognizing different epitopes can help characterize the nature of specific mutations

  • Compare results with wild-type controls from healthy individuals

• Compensatory mechanisms:

  • Investigate RAB27B upregulation in GS2 patients using specific antibodies to each isoform

  • Assess correlation between RAB27B compensation and disease severity

• Therapeutic research:

  • Monitor restoration of RAB27A expression in gene therapy approaches

  • Evaluate changes in downstream effector pathways following therapeutic interventions

• Cell-specific effects:

  • Compare RAB27A expression and localization across different cell types affected in GS2 (melanocytes, cytotoxic T cells, natural killer cells)

  • Correlate expression patterns with cell-specific functional defects

How are RAB27A antibodies being used to investigate extracellular vesicle biogenesis and function?

Extracellular vesicle (EV) research represents a frontier where RAB27A antibodies are providing valuable insights:

• EV isolation verification: RAB27A antibodies help confirm successful isolation of specific EV subpopulations, as RAB27A is enriched in certain exosome preparations. Exosome Standards (K562) serve as validated positive controls for RAB27A detection in EV research .

• Mechanistic studies of EV biogenesis:

  • Immunofluorescence co-localization studies with RAB27A antibodies and multivesicular body markers

  • Western blot analysis of sucrose gradient fractions to identify RAB27A-positive vesicle populations

  • Immunoprecipitation to identify RAB27A effector proteins in the EV secretion pathway

• Cancer EV research: In colorectal cancer and melanoma models, RAB27A antibodies help track changes in EV secretion mechanisms that may contribute to tumor progression or drug resistance .

• Quantitative approaches:

  • Use Simple Western systems for automated, quantitative analysis of RAB27A in EV preparations

  • Flow cytometry of EVs using bead-based capture systems and RAB27A antibodies

• EV cargo sorting: Investigate RAB27A's role in determining EV cargo content through knockdown studies monitored by RAB27A antibodies.

What approaches can resolve contradictory findings about RAB27A expression in cancer tissues?

The literature contains contradictory findings regarding RAB27A expression and its prognostic significance in cancers like colorectal cancer:

• Standardized antibody validation:

  • Use antibodies validated with knockout/knockdown controls

  • Implement consistent staining protocols across studies

  • Consider multiple antibodies targeting different epitopes to confirm findings

• Comprehensive sampling strategies:

  • Increase sample size to minimize statistical errors (studies with 388 samples showed more consistent results than those with 112 samples)

  • Include diverse patient populations and tumor stages

  • Use tissue microarrays for higher throughput and standardized processing

• Multi-omics integration:

  • Correlate protein expression (immunohistochemistry with RAB27A antibodies) with mRNA expression (qPCR, RNA-seq)

  • Consider post-translational modifications using phospho-specific antibodies if available

  • Integrate with genomic data to identify mutations or copy number alterations

• Functional stratification:

  • Assess RAB27A in context of its interacting partners

  • Consider cancer subtype-specific effects

  • Evaluate RAB27A in relation to specific cellular processes (exosome secretion, migration, etc.)

• Meta-analysis approach:

  • Systematically review existing studies with attention to methodological differences

  • Develop standardized reporting guidelines for RAB27A expression studies

  • Pool data when possible to increase statistical power

How can RAB27A antibodies contribute to understanding the role of RAB27A in insulin secretion and diabetes research?

RAB27A plays an important role in insulin secretion from pancreatic β-cells, making it relevant for diabetes research:

• Subcellular localization studies:

  • Immunofluorescence with RAB27A antibodies to visualize its localization on mature insulin granules in β-cells

  • Co-localization with insulin and other granule markers

  • Live-cell imaging using fluorescently-tagged antibody fragments to track RAB27A dynamics during glucose stimulation

• Expression analysis in disease models:

  • Western blot quantification of RAB27A levels in islets from diabetic vs. non-diabetic models

  • Immunohistochemistry of pancreatic sections from patients with different forms of diabetes

  • Correlation of RAB27A expression with insulin secretion capacity

• Mechanistic investigations:

  • Immunoprecipitation with RAB27A antibodies to identify interaction partners in β-cells

  • Analysis of RAB27A activation state during biphasic insulin secretion

  • Investigation of RAB27A post-translational modifications in response to glucose

• Therapeutic implications:

  • Monitoring RAB27A expression and localization following treatment with anti-diabetic agents

  • Screening for compounds that modulate RAB27A activity as potential therapeutic approach

  • Evaluation of RAB27A as a biomarker for β-cell dysfunction