HPS6 Antibody

What is HPS6 and why is it studied?

HPS6 (Hermansky-Pudlak Syndrome 6) is a protein involved in a rare autosomal recessive disorder characterized by ocular albinism (OA) or oculocutaneous albinism (OCA) and platelet dysfunction. It plays a critical role in the biogenesis of lysosome-related organelles including melanosomes and platelet-dense granules. Recent research has expanded our understanding of HPS6's involvement in cellular trafficking pathways and organelle formation, making it an important target for both basic research and clinical investigations . The protein has a calculated molecular weight of 775 amino acids (83 kDa) and is encoded by the gene with NCBI ID 79803 .

What applications are HPS6 antibodies validated for?

HPS6 antibodies have been validated for multiple experimental applications including:

Optimal dilutions should be determined experimentally for each specific application and sample type .

What species reactivity can be expected from commercially available HPS6 antibodies?

Available HPS6 antibodies demonstrate confirmed reactivity with human, mouse, and rat samples. Published studies have primarily cited reactivity with human and mouse tissues . When selecting an antibody for your research, verify the specific reactivity profiles as they may vary between manufacturers and antibody clones. Polyclonal antibodies raised against conserved regions may show cross-reactivity across multiple species, while those targeting variable regions may be more species-specific .

What are the recommended protocols for antigen retrieval when using HPS6 antibodies in IHC?

For optimal immunohistochemical detection of HPS6, two antigen retrieval methods have proven effective:

• Primary recommended method: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

The choice between these methods should be determined experimentally based on your specific tissue type and fixation protocol. Proper antigen retrieval is critical for accessing epitopes that may be masked during fixation processes, particularly in formalin-fixed, paraffin-embedded (FFPE) samples . Following antigen retrieval, standard blocking and antibody incubation protocols should be followed with the recommended dilution ranges (1:50-1:500) for IHC applications.

How should I optimize Western blot protocols for detecting HPS6?

For optimal Western blot detection of HPS6:

• Sample preparation: Use cell lines with confirmed HPS6 expression (HeLa, HEK-293, or MCF-7 cells) as positive controls

• Loading amount: Start with 20-30 μg of total protein per lane

• Separation: Use 8-10% SDS-PAGE gels for optimal resolution of the 83 kDa protein

• Transfer conditions: Semi-dry or wet transfer at 100V for 60-90 minutes

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Start with 1:500 dilution in blocking buffer and optimize as needed

• Incubation: Overnight at 4°C with gentle rocking

• Detection: Compatible secondary antibody systems followed by standard chemiluminescence or fluorescence detection

The observed molecular weight should be approximately 83 kDa, which matches the calculated molecular weight based on the 775 amino acid sequence .

What controls should be included when using HPS6 antibodies in research applications?

Robust experimental design requires appropriate controls when using HPS6 antibodies:

• Positive tissue/cell controls: HeLa, HEK-293, or MCF-7 cells have confirmed HPS6 expression

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at matching concentration)

  • Tissue known to lack HPS6 expression

• Knockdown/knockout validation: Where available, HPS6 KD/KO samples provide the strongest specificity control (published validations exist for HPS6 knockdown)

• Blocking peptide control: Pre-incubation of antibody with immunizing peptide should abolish specific staining

These controls help distinguish specific from non-specific signals and validate antibody performance in your experimental system.

How can HPS6 antibodies be used to study the pathophysiology of Hermansky-Pudlak Syndrome?

HPS6 antibodies serve as valuable tools for investigating the underlying mechanisms of Hermansky-Pudlak Syndrome:

• Protein localization studies: IHC and ICC can reveal altered subcellular distribution of HPS6 in patient samples

• Functional complex analysis: IP followed by mass spectrometry can identify disrupted protein interactions in HPS patient cells

• Quantitative analysis: Western blotting can determine if specific mutations affect protein expression levels

• Trafficking studies: Dual immunofluorescence with markers for melanosomes, platelet-dense granules, or lysosomes can reveal defects in organelle biogenesis

• Genotype-phenotype correlations: Antibody-based assays can connect specific HPS6 variants to functional outcomes

Recent studies have identified novel variants in HPS6 causing suspected ocular albinism in Chinese families, illustrating how molecular diagnostics can refine clinical classification . These variants (c.1674dup, c.503-504del, c.1114C>T, and c.1556del) can be studied at the protein level using specific antibodies to understand their impact on protein expression, localization, and function.

What is the relationship between HPS6 and Weibel-Palade Body biogenesis?

Recent research has revealed that HPS6 plays a regulatory role in the biogenesis of Weibel-Palade Bodies (WPBs), which are specialized organelles in endothelial cells that store von Willebrand factor and other proteins involved in hemostasis and inflammation .

HPS6 likely regulates WPB biogenesis by participating in the trafficking of v-ATPase to the WPB membrane . This finding expands our understanding of HPS6 beyond its established role in melanosome and platelet dense granule formation. Researchers investigating this relationship should consider:

• Co-immunoprecipitation studies using HPS6 antibodies to identify interactions with v-ATPase components

• Immunofluorescence co-localization studies of HPS6 with WPB markers

• Live-cell imaging using tagged HPS6 to track its role in WPB formation

• Functional studies examining how HPS6 depletion affects WPB maturation and acidification

These approaches can further elucidate the mechanistic details of HPS6's function in this context.

How can HPS6 antibodies be used to investigate DNA methylation impacts on HPS6 expression?

Recent research has revealed a significant overlap between CpG islands and variants of HPS6, suggesting a potential link between DNA methylation and HPS6 variants . To investigate this relationship:

• Chromatin immunoprecipitation (ChIP) assays: Use HPS6 antibodies alongside methylation-specific antibodies to examine the relationship between methylation status and HPS6 binding to chromatin

• Expression correlation studies: Compare HPS6 protein levels (via Western blot) with methylation status of the HPS6 promoter

• Epigenetic modulation experiments: Treat cells with DNA methyltransferase inhibitors and assess changes in HPS6 expression

• Patient-derived sample analysis: Compare methylation patterns with HPS6 protein expression in samples from individuals with different HPS6 variants

This approach could provide insights into epigenetic regulation of HPS6 and potentially reveal new therapeutic targets for Hermansky-Pudlak Syndrome.

What are common issues when using HPS6 antibodies in IHC and how can they be resolved?

Researchers may encounter several challenges when using HPS6 antibodies for immunohistochemistry:

For human tissue samples specifically, it's recommended to first try antigen retrieval with TE buffer pH 9.0, as this has been validated for human ovary tumor tissue and kidney tissue .

How should discrepancies in observed molecular weight be interpreted when using HPS6 antibodies?

While the calculated molecular weight of HPS6 is 83 kDa (based on its 775 amino acid sequence), researchers may observe variations in apparent molecular weight on Western blots . These discrepancies may be attributed to:

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications can alter protein migration

• Alternative splicing: Different isoforms may be expressed in different tissues

• Proteolytic processing: HPS6 may undergo cleavage during sample preparation

• Technical factors: Gel percentage, running conditions, and marker systems can affect apparent molecular weight

To address these issues:

• Use positive control samples with known HPS6 expression (HeLa, HEK-293, MCF-7 cells)

• Consider running samples from multiple tissues to identify tissue-specific variations

• Include detailed methods reporting apparent molecular weight in publications

• When possible, confirm identity through additional methods (mass spectrometry, immunoprecipitation)

What new HPS6 variants have been discovered and how can antibodies help characterize them?

Recent research has identified previously unreported HPS6 variants associated with suspected ocular albinism:

These variants expand the spectrum of known HPS6 mutations. HPS6 antibodies can help characterize these variants by:

• Assessing protein expression levels to determine if truncated proteins are produced and stable

• Examining subcellular localization patterns of mutant proteins

• Evaluating the impact on protein-protein interactions through co-immunoprecipitation studies

• Correlating protein expression with phenotypic severity

These molecular insights can improve diagnostic accuracy and potentially inform therapeutic approaches for HPS6-related disorders .

How do HPS6 variants correlate with clinical phenotypes in Hermansky-Pudlak Syndrome?

Interestingly, recent comprehensive analysis of HPS6 variants and their associated phenotypes has not identified significant genotype-phenotype correlations . Analysis of 46 probands from families or sporadic cases with pathogenic HPS6 variants revealed:

• No clear correlation between the specific HPS6 variant and disease severity

• No observable relationship between the truncation length of the HPS6 protein and phenotypic manifestations

• Spatial aggregation of variants in the 3D structure of HPS6 protein, suggesting functional significance of certain structural regions

These findings have important implications for researchers using HPS6 antibodies to study disease mechanisms:

• Focus on identifying functional domains affected by mutations

• Study the impact of variants on protein-protein interactions

• Investigate tissue-specific effects of different mutations

• Consider environmental or genetic modifiers that may influence phenotypic expression

Continued research with HPS6 antibodies will be crucial for understanding the complex pathophysiology of this syndrome and identifying potential therapeutic targets .

What are the optimal storage conditions for HPS6 antibodies?

For maximum stability and activity retention of HPS6 antibodies:

• Long-term storage: Store at -20°C in manufacturers' recommended buffer

• Working dilutions: Prepare fresh or store at 4°C for up to one week

• Freeze-thaw cycles: Minimize by preparing single-use aliquots

• Buffer conditions: PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) is typically used

How should I validate a new lot of HPS6 antibody before using it in critical experiments?

When receiving a new lot of HPS6 antibody, validation is essential to ensure experimental reproducibility:

• Western blot comparison: Run the previous and new lots side-by-side using positive control samples (HeLa, HEK-293, or MCF-7 cells)

• Titration experiment: Test a range of dilutions to determine optimal working concentration

• Specificity check: Confirm single band at expected molecular weight (83 kDa)

• Application validation: Test in your specific application (WB, IHC, IP) with appropriate controls

• Performance documentation: Record lot number, dilution, and performance characteristics for future reference