HRASLS5 Antibody

What is HRASLS5 and what cellular functions does it perform?

HRASLS5 (also known as PLAAT5, HRLP5, HRSL5, or Ca(2+)-independent N-acyltransferase) is a member of the HRASLS family known for its dual enzymatic functionality. It exhibits both phospholipase A1/2 and acyltransferase activities, catalyzing the calcium-independent release of fatty acids from glycerophospholipids and the transfer of fatty acyl groups from phosphatidylcholine to phosphatidylethanolamine . These activities are crucial for lipid metabolism and cell signaling pathways. HRASLS5 has been implicated in cancer progression, neurodegenerative diseases, and various metabolic disorders, making it a target of considerable research interest .

What tissue types express HRASLS5 protein?

HRASLS5 expression has been detected in multiple human tissues. Immunohistochemistry studies have confirmed its presence in human placenta, heart, ovary, skin, and spleen tissues . Additionally, research in animal models has shown that HRASLS5 is primarily expressed in spermatocytes within the maturing rat testis, suggesting a potential role in spermatogenesis . The varied tissue distribution indicates that HRASLS5 likely performs different physiological functions depending on the cellular context.

What applications are HRASLS5 antibodies suitable for?

HRASLS5 antibodies have been validated for several research applications:

When selecting an antibody, researchers should consider the specific application requirements and choose an antibody validated for that particular technique .

What are the recommended dilutions for HRASLS5 antibodies?

Optimal dilutions vary depending on both the antibody and application:

It is strongly recommended to perform a titration experiment for each new sample type or application to determine the optimal antibody concentration. The dilution may need adjustment based on sample type, detection method, and instrumentation sensitivity .

How should HRASLS5 antibodies be stored for optimal stability?

Most HRASLS5 antibodies are supplied in a liquid form with stabilizing agents. For example:

• Antibody 17581-1-AP is provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 and should be stored at -20°C

• STJA0008449 comes in PBS containing 50% Glycerol, 0.5% BSA, and 0.02% Sodium Azide and should be stored at -20°C

• PACO09795 is supplied in PBS (pH 7.4) containing 0.02% sodium azide and 50% glycerol

To maintain antibody integrity, avoid repeated freeze-thaw cycles. For antibodies supplied in larger volumes, consider aliquoting upon receipt to minimize freeze-thaw events. Most antibodies remain stable for approximately one year when stored properly .

How can researchers validate the specificity of HRASLS5 antibodies in their experimental systems?

Validating antibody specificity is crucial for reliable results. Consider these methodological approaches:

• Positive and negative control tissues: Based on known expression patterns, use human placenta, heart, or testis tissues as positive controls. Include tissues with negligible HRASLS5 expression as negative controls .

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide prior to application. Diminished signal indicates specificity for the target epitope.

• siRNA knockdown validation: Treat cells with HRASLS5-specific siRNA and confirm reduced antibody signal by Western blot or immunocytochemistry compared to non-targeting siRNA controls.

• Recombinant protein controls: Use purified HRASLS5 protein as a positive control in Western blot applications to confirm antibody recognition of the target at the correct molecular weight (approximately 29 kDa) .

• Cross-validate with multiple antibodies: Employ antibodies targeting different epitopes of HRASLS5 to confirm consistent localization or expression patterns.

What are the key considerations when designing immunohistochemistry experiments with HRASLS5 antibodies?

Successful IHC with HRASLS5 antibodies requires careful optimization:

• Antigen retrieval optimization: For antibody 17581-1-AP, suggested antigen retrieval uses TE buffer at pH 9.0, though citrate buffer at pH 6.0 may serve as an alternative . Compare both methods to determine optimal conditions for your specific tissue samples.

• Fixation considerations: Overfixation can mask epitopes. If using FFPE tissues, standardize fixation times (typically 24 hours in 10% neutral buffered formalin) for consistent results.

• Blocking optimization: Due to HRASLS5's involvement in lipid metabolism, lipid-rich tissues may exhibit higher background. Use 5-10% normal serum from the same species as the secondary antibody, along with 0.1-0.3% Triton X-100 for enhanced blocking.

• Positive control inclusion: Include human placenta, heart, or ovary tissue sections as positive controls when optimizing staining protocols .

• Signal amplification: For tissues with low HRASLS5 expression, consider using polymer-based detection systems or tyramide signal amplification to enhance sensitivity without increasing background.

How does HRASLS5 expression change during disease progression, and what methodological approaches are best for tracking these changes?

HRASLS5 has been implicated in cancer progression and metabolic disorders, suggesting expression changes during pathological states . For comprehensive analysis:

• Tissue microarrays (TMAs): Analyze HRASLS5 expression across multiple patient samples representing different disease stages using standardized IHC protocols with the 17581-1-AP antibody at 1:50 dilution .

• Quantitative immunoblotting: Use densitometry analysis of Western blots with STJA0008449 antibody (1:1000) to quantify HRASLS5 protein levels, normalizing to housekeeping proteins like GAPDH or β-actin.

• Multiplex immunofluorescence: Co-stain for HRASLS5 alongside disease markers to assess spatial relationships and expression correlations during disease progression.

• Longitudinal sampling: In animal models or patient-derived samples, collect tissues at multiple timepoints to track HRASLS5 expression changes throughout disease development.

• Functional correlation: Combine expression analysis with enzymatic activity assays to determine whether HRASLS5's phospholipase and acyltransferase activities correlate with expression changes during disease.

What are potential cross-reactivity issues with HRASLS5 antibodies and other HRASLS family members?

The HRASLS family contains several homologous proteins that may share epitopes with HRASLS5, potentially leading to cross-reactivity:

• Sequence homology analysis: Before selecting an antibody, analyze the immunogen sequence for homology with other HRASLS family members. Choose antibodies raised against unique regions of HRASLS5.

• Western blot validation: Perform Western blot analysis using recombinant proteins for multiple HRASLS family members to assess potential cross-reactivity.

• Knockout controls: When available, utilize HRASLS5 knockout cell lines or tissues as negative controls to confirm antibody specificity.

• Epitope mapping: Consider epitope mapping studies to identify the exact binding region of the antibody and compare it with potential cross-reactive sites in related proteins.

• Pre-absorption controls: Pre-absorb the antibody with recombinant proteins of related family members to determine if this affects staining patterns in your samples.

How can researchers troubleshoot inconsistent HRASLS5 antibody staining in tissue samples?

Inconsistent staining can result from various factors:

• Sample preparation variability: Standardize fixation protocols and processing times. For FFPE tissues, excessive fixation can mask epitopes, while inadequate fixation may result in tissue degradation.

• Antigen retrieval optimization: For HRASLS5 detection, compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) . Optimize retrieval time and temperature based on your specific tissue type.

• Antibody titration: Perform systematic titrations (e.g., 1:20, 1:50, 1:100, 1:200) to identify the optimal concentration that maximizes specific signal while minimizing background .

• Detection system comparison: If using chromogenic detection, compare DAB versus AEC substrates. For fluorescent detection, evaluate directly conjugated antibodies versus secondary antibody approaches.

• Tissue heterogeneity considerations: HRASLS5 expression may vary within tissues. Consider using larger tissue sections or multiple cores in TMAs to account for heterogeneity.

• Batch effects management: Process all comparative samples simultaneously with the same reagent lots. Include a standard reference sample in each batch to normalize for inter-batch variation.

What enzymatic functions of HRASLS5 might influence antibody selection and experimental design?

HRASLS5 exhibits dual enzymatic activities that may affect experimental approaches:

• Conformational considerations: HRASLS5's phospholipase A1/2 and acyltransferase activities may involve conformational changes that alter epitope accessibility. Consider antibodies targeting different regions for comprehensive analysis.

• Activity-state specific detection: Some epitopes may be accessible only when HRASLS5 is in specific functional states. When studying enzymatic activities, use multiple antibodies targeting different regions.

• Substrate interactions: HRASLS5's interaction with lipid substrates may mask certain epitopes. For co-localization studies with lipid substrates, select antibodies whose epitopes remain accessible during substrate binding.

• Experimental conditions influence: The calcium-independent nature of HRASLS5's enzymatic activities suggests that chelating agents in buffers won't affect its conformation, but detergents used for cell lysis may disrupt lipid-protein interactions critical for native structure.

• Post-translational modifications: Consider whether phospholipase or acyltransferase activity depends on post-translational modifications that might affect antibody binding.

What are the recommended controls when using HRASLS5 antibodies in research?

Rigorous controls ensure reliable results with HRASLS5 antibodies:

• Positive tissue controls: Include human placenta, heart, ovary, skin, or spleen tissues, which have demonstrated HRASLS5 expression .

• Negative controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG at the same concentration)

  • Non-expressing tissues or cells

• Knockdown/knockout validation: When available, include HRASLS5 siRNA-treated or CRISPR-edited samples to confirm specificity.

• Peptide competition: Pre-incubate antibody with immunogenic peptide to confirm binding specificity.

• Multiple antibody validation: When critical findings depend on HRASLS5 detection, confirm with at least two antibodies targeting different epitopes.

• Recombinant protein control: Include purified HRASLS5 protein (29 kDa) as a positive control in Western blot applications .

How can HRASLS5 antibodies be used to investigate its role in cancer biology and therapeutic development?

HRASLS5 has been implicated in cancer progression , offering several research directions:

• Expression profiling: Use 17581-1-AP antibody for IHC analysis of tumor microarrays to correlate HRASLS5 expression with clinical outcomes across multiple cancer types .

• Signaling pathway analysis: Combine HRASLS5 antibodies with phospho-specific antibodies for RAS pathway components to elucidate HRASLS5's role in modulating oncogenic signaling.

• Functional studies: Following genetic manipulation of HRASLS5 levels, use antibodies to confirm expression changes before assessing effects on proliferation, migration, and invasion.

• Drug response biomarker: Evaluate HRASLS5 expression before and after treatment with therapeutics targeting related pathways to assess its potential as a response biomarker.

• Therapeutic antibody development: Investigate whether HRASLS5 has accessible epitopes on the cell surface of cancer cells that could be targeted with therapeutic antibodies.

• Lipid metabolism connections: Study how HRASLS5's enzymatic activities influence cancer cell metabolism by analyzing expression in conjunction with lipid profiling.

What future research directions might benefit from improved HRASLS5 antibodies?

While current HRASLS5 antibodies serve many research applications, several emerging areas would benefit from further antibody development:

• Isoform-specific antibodies: Development of antibodies that can distinguish between potential HRASLS5 splice variants or isoforms would enable more precise functional studies.

• Phospho-specific antibodies: Creation of antibodies recognizing phosphorylated forms of HRASLS5 would facilitate studies of its regulation through post-translational modifications.

• Activity-state specific antibodies: Antibodies that specifically recognize active versus inactive conformations would advance understanding of HRASLS5's enzymatic functions in different cellular contexts.

• Super-resolution microscopy-compatible antibodies: Directly conjugated antibodies optimized for super-resolution techniques would allow detailed subcellular localization studies.

• Therapeutic applications: Further characterization of HRASLS5's role in disease might identify opportunities for therapeutic antibody development targeting this protein in specific pathological contexts.