RAB27A Antibody, Biotin conjugated

What is RAB27A and what cellular functions is it involved in?

RAB27A is a member of the RAS oncogene family, specifically a small GTPase that functions as a key regulator of organelle movement and regulated exocytosis in secretory cells. It plays crucial roles in vesicle trafficking and membrane fusion events. RAB27A is particularly important in the secretion of dense granules in platelets, although it works somewhat redundantly with RAB27B in this context . Mutations in the RAB27A gene in humans result in Griscelli syndrome, primarily affecting melanocytes and cytotoxic T lymphocytes . RAB27A is also involved in the regulation of exosome secretion pathways and plays important roles in various cellular processes requiring precise vesicular trafficking .

What are the recommended storage conditions for RAB27A Antibody, Biotin conjugated?

For optimal stability and performance, RAB27A Antibody, Biotin conjugated should be stored at -20°C in aliquots to avoid repeated freeze-thaw cycles . The antibody is typically shipped in a liquid form containing a buffer of 0.01 M PBS (pH 7.4) with 0.03% Proclin-300 and 50% glycerol . It's important to protect the antibody from prolonged exposure to light due to the biotin conjugation, as this can affect the stability of the conjugate . For short-term storage during experimental work, the antibody can be kept at 4°C for up to one month, but should be returned to -20°C for long-term storage .

How can I optimize the detection of RAB27A in immunohistochemistry experiments?

While the biotin-conjugated antibody is primarily recommended for ELISA, optimization for IHC can be approached based on protocols used with other RAB27A antibody formats. Based on validated protocols, heat-mediated antigen retrieval in citrate buffer (pH 6) for 20 minutes is recommended for paraffin-embedded tissue sections . Blocking with 10% goat serum reduces non-specific binding . For optimal results with the biotin-conjugated antibody, researchers should:

• Perform titration experiments to determine optimal antibody concentration (starting with 1μg/ml based on successful protocols for non-conjugated versions)

• Incubate sections with primary antibody overnight at 4°C

• Use streptavidin-HRP or another streptavidin-conjugated detection system directly (no secondary antibody needed)

• Develop using DAB or another appropriate chromogen

• Include appropriate positive control tissues such as lung cancer or mammary cancer tissue, which have shown strong RAB27A expression

Successful staining protocols have demonstrated RAB27A detection in human lung cancer, mammary cancer, rat small intestine, and rat spleen tissues, suggesting these as appropriate positive controls .

What is the relationship between RAB27A and RAB27B, and how might this impact experimental design?

RAB27A and RAB27B are closely related homologs within the Rab27 GTPase subfamily. While they share significant sequence similarity, they demonstrate both redundant and non-redundant functions. Key considerations for experimental design include:

• Functional overlap: Both proteins participate in dense granule secretion in platelets, with RAB27A partially compensating for the absence of RAB27B .

• Differential expression: RAB27B is more restrictedly expressed compared to the widely expressed RAB27A . Studies in platelets showed lower RAB27A expression compared to RAB27B .

• Distinct roles: While both participate in secretion, RAB27B appears to have non-redundant functions in dense granule formation or "packaging" that cannot be compensated by RAB27A .

This relationship necessitates careful experimental design when studying either protein. When investigating RAB27A function, researchers should consider:

• Including RAB27B detection controls to account for potential compensation effects

• Using knockdown/knockout systems for both proteins to distinguish unique versus redundant functions

• Choosing appropriate cell types where one isoform may predominate over the other

• Designing experiments that can distinguish between formation/packaging roles versus secretory roles

How can I validate the specificity of RAB27A Antibody, Biotin conjugated in my experimental system?

Validating antibody specificity is crucial for reliable experimental results. For RAB27A Antibody, Biotin conjugated, consider the following validation approaches:

• Positive and negative tissue controls: Compare staining in tissues known to express RAB27A (e.g., human lung cancer tissue, mammary cancer tissue, rat small intestine, rat spleen) versus tissues with low or no expression .

• siRNA knockdown validation: Perform experiments in cells with and without RAB27A knockdown to confirm specificity of signal reduction.

• Peptide competition assay: Pre-incubate the antibody with purified RAB27A protein or immunogenic peptide before applying to samples. Specific staining should be blocked.

• Testing in Rab27a knockout models: Validate absence of signal in available Rab27a knockout models (e.g., ashen mice) .

• Cross-reactivity assessment: Test for potential cross-reactivity with RAB27B using RAB27B-overexpressing systems or in tissues with differential expression of the two proteins.

• Western blot analysis: Confirm detection of a single band of appropriate molecular weight (approximately 25 kDa for RAB27A).

• Comparison with alternative antibody clones: Validate results using alternative antibodies targeting different epitopes of RAB27A.

Documentation of these validation steps substantially strengthens the reliability of experimental results, particularly in research focusing on the specific roles of RAB27A versus related proteins.

How can I use RAB27A Antibody, Biotin conjugated to investigate the role of RAB27A in exosome secretion pathways?

Investigating RAB27A in exosome secretion requires specialized experimental approaches. While the biotin-conjugated antibody is primarily validated for ELISA, it can be adapted for exosome research through multiple approaches:

• Co-localization studies by confocal microscopy:

  • Use the biotin-conjugated antibody with streptavidin-fluorophore detection

  • Co-stain with markers for multivesicular bodies (MVBs) like CD63

  • Visualize RAB27A localization during exosome biogenesis and secretion

  • Quantify co-localization with exosome markers at different stages

• Immunoprecipitation of RAB27A-associated complexes:

  • Use the biotin-conjugated antibody with streptavidin magnetic beads

  • Pull down RAB27A and associated proteins

  • Analyze the complex by mass spectrometry to identify novel interaction partners

  • Compare protein associations in different cell types or under various stimulation conditions

• ELISA-based quantification of RAB27A in exosome fractions:

  • Isolate exosomes using differential ultracentrifugation or size exclusion chromatography

  • Develop a sandwich ELISA using the biotin-conjugated antibody

  • Quantify RAB27A content in exosome fractions versus cellular fractions

  • Compare RAB27A levels across different experimental conditions

• Proximity ligation assay (PLA):

  • Use the biotin-conjugated antibody with another antibody targeting a potential interaction partner

  • Visualize and quantify protein interactions in situ

  • Map the spatial and temporal dynamics of RAB27A interactions during exosome biogenesis

When designing these experiments, it's important to consider that RAB27A function may vary between cell types, with particular importance in secretory cells where regulated exocytosis is a primary function .

What techniques can be used to investigate the interaction between RAB27A and its effector proteins using biotin-conjugated antibodies?

The biotin-conjugated RAB27A antibody provides flexibility for studying protein-protein interactions through several sophisticated approaches:

• Pull-down assays with nucleotide-locked RAB27A mutants:

  • Create GTP-locked (constitutively active) and GDP-locked (inactive) RAB27A mutants

  • Perform pull-downs using biotin-conjugated antibody and streptavidin beads

  • Compare effector binding between active and inactive states

  • Analyze by western blot or mass spectrometry

• Proximity-dependent biotin identification (BioID):

  • Generate fusion constructs of RAB27A with a biotin ligase

  • Express in cells of interest and provide biotin

  • Identify biotinylated proteins (proximal to RAB27A) using the biotin-conjugated antibody

  • Map the RAB27A proximal proteome in different cellular compartments

• FRET-based interaction studies:

  • Use biotin-conjugated RAB27A antibody with streptavidin-fluorophore

  • Pair with fluorescently labeled effector proteins

  • Measure FRET signal as indication of protein proximity

  • Track dynamic interactions in live cells

• Biolayer interferometry (BLI) or surface plasmon resonance (SPR):

  • Immobilize biotin-conjugated RAB27A antibody on streptavidin sensors

  • Capture RAB27A from cell lysates

  • Measure binding kinetics with purified effector proteins

  • Determine affinity constants for different interactions

When investigating these interactions, it's important to consider that RAB27A functions within a complex network of proteins. Studies in platelets have shown that RAB27A can partially compensate for RAB27B in secretion processes, suggesting overlapping effector interactions, while other RAB27B functions appear non-redundant . This implies the existence of both shared and unique effector proteins for these related GTPases.

What are the considerations when using RAB27A Antibody, Biotin conjugated in studies of diseases associated with RAB27A dysfunction?

When investigating diseases associated with RAB27A dysfunction, such as Griscelli syndrome, several important considerations should guide experimental design:

• Mutation-specific effects on epitope recognition:

  • Different mutations in RAB27A may alter antibody epitope availability

  • Verify antibody recognition of mutant forms using recombinant proteins

  • Consider using multiple antibodies targeting different epitopes

  • Document the specific immunogen sequence used for antibody production

• Expression level versus functionality assessment:

  • Diseased states may show normal protein levels but impaired function

  • Complement antibody detection with functional assays

  • Assess RAB27A GTP binding, hydrolysis, and effector interactions

  • Correlate protein detection with functional readouts

• Tissue-specific effects in disease models:

  • RAB27A mutations primarily affect melanocytes and cytotoxic T lymphocytes in Griscelli syndrome

  • Verify antibody performance in disease-relevant tissues

  • Include appropriate disease model tissues as controls

  • Consider the differential expression of compensatory proteins (like RAB27B) across tissues

• Compensation mechanisms:

  • Assess potential upregulation of RAB27B or other related proteins

  • Design experiments to distinguish between redundant functions

  • Consider the viability of double knockout models when interpreting single protein defects

• Detection in patient-derived samples:

  • Optimize protocols for clinical specimens

  • Account for potential heterogeneity in patient samples

  • Include appropriate controls from both healthy donors and disease models

  • Standardize sample collection and processing

Research on Rab27 knockout mice has demonstrated that specific cell types rely differentially on single Rab27 isoforms, with melanocytes and cytotoxic T lymphocytes depending primarily on Rab27a, while platelets depend more on Rab27b . This selective dependency should inform the experimental approach when studying diseases with RAB27A dysfunction.

What are the optimal dilution ranges for RAB27A Antibody, Biotin conjugated across different applications?

While the manufacturer recommends that optimal dilutions should be determined by the end user , starting points based on related antibody formats and applications can be suggested:

Always perform titration experiments to determine the optimal concentration for your specific experimental system. The antibody has demonstrated reactivity with human samples, and non-conjugated formats have shown cross-reactivity with mouse and rat tissues . When optimizing dilutions, consider the following:

• Start with the middle of the recommended range

• Test at least 3-5 different dilutions in a geometric series

• Include appropriate positive and negative controls

• Evaluate signal-to-noise ratio rather than absolute signal intensity

• Consider the detection system sensitivity when determining optimal dilution

How can I troubleshoot weak or non-specific signals when using RAB27A Antibody, Biotin conjugated?

When encountering issues with signal strength or specificity, consider the following troubleshooting approaches:

For weak signals:

• Antibody concentration: Increase the concentration of primary antibody within recommended ranges.

• Antigen retrieval: For IHC/ICC applications, optimize antigen retrieval methods. Heat-mediated antigen retrieval in citrate buffer (pH6) for 20 minutes has been validated for RAB27A detection .

• Incubation conditions: Extend incubation time (e.g., overnight at 4°C) or adjust temperature.

• Detection system: Enhance sensitivity using amplification systems like tyramide signal amplification (TSA).

• Buffer optimization: Test different blocking buffers to reduce background while preserving specific signal.

• Sample preparation: Ensure tissue fixation and processing are optimal for preserving the target epitope.

For non-specific signals:

• Blocking optimization: Increase blocking time or concentration (10% goat serum has been validated) .

• Antibody dilution: Increase dilution to reduce non-specific binding.

• Washing stringency: Increase number or duration of wash steps.

• Cross-reactivity assessment: Test for potential cross-reactivity with RAB27B using appropriate controls.

• Absorption controls: Pre-absorb the antibody with recombinant RAB27A protein to confirm signal specificity.

• Secondary reagent specificity: Ensure streptavidin reagents are not binding non-specifically to endogenous biotin.

Sample-specific considerations:

• Endogenous biotin blocking: For biotin-rich tissues (liver, kidney, brain), use avidin/biotin blocking kits before applying the biotin-conjugated primary antibody.

• Endogenous peroxidase quenching: For IHC applications, ensure adequate quenching of endogenous peroxidase activity.

• Tissue-specific autofluorescence: For fluorescent detection, use appropriate autofluorescence reduction methods.

Successful RAB27A detection has been documented in various tissues including human lung cancer, mammary cancer, rat small intestine, and rat spleen , providing valuable positive control references.