SHISA5 Antibody

What is SHISA5 and what cellular pathways is it involved in?

SHISA5 (also known as Scotin) functions as a pro-apoptotic protein that operates in a caspase-dependent manner, playing a significant role in p53/TP53-dependent apoptosis . The protein is encoded by the SHISA5 gene (Gene ID: 51246) and has a molecular weight of approximately 25.6 kDa . Several isoforms of SHISA5 have been identified, as evidenced by multiple NCBI accession numbers (NP_001258994, NP_001258995, NP_001258996, NP_001258997, NP_001259011, NP_001259012, NP_057563) . Understanding SHISA5's role in apoptotic pathways is essential for cancer research and cell death studies.

What types of SHISA5 antibodies are available for research applications?

SHISA5 antibodies are available in several configurations, differentiated by their binding specificity, host organism, clonality, and conjugation status:

Selection should be based on your specific experimental needs, target epitope, and intended application .

How should I determine the optimal dilution ratios for different SHISA5 antibody applications?

Optimal dilution ratios vary by application and specific antibody. For SHISA5 antibodies targeting AA 75-102, a 1:1000 dilution is recommended for Western blotting . For antibodies targeting AA 56-137, the recommended dilutions are: IHC (1:20-1:200), IF (1:50-1:500), and IP (1:200-1:2000) . These ratios should be considered starting points, and optimization is necessary for each experimental system. A titration experiment is recommended, where a range of antibody concentrations are tested against the same sample amount. The optimal dilution provides the best signal-to-noise ratio with minimal background staining while conserving antibody .

What are the proper storage conditions for maintaining SHISA5 antibody activity?

Storage conditions for SHISA5 antibodies vary by formulation. For the AA 75-102 antibody, refrigeration at 2-8°C is suitable for up to 6 months, while long-term storage requires -20°C . The AA 56-137 antibody should be stored at -20°C or -80°C, and repeated freeze-thaw cycles should be avoided . Most SHISA5 antibodies are supplied in a liquid format with specific buffer systems (e.g., PBS with 0.09% sodium azide or 50% glycerol with 0.01M PBS pH 7.4) . Proper aliquoting of antibodies upon receipt can minimize freeze-thaw cycles and maintain antibody integrity over longer periods .

How can I validate SHISA5 antibody specificity for experiments studying p53-dependent apoptosis?

Validating SHISA5 antibody specificity is critical when studying p53-dependent apoptosis. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines with known SHISA5 expression levels. HCT116 p53+/+ and p53-/- isogenic cell lines are ideal for comparing SHISA5 expression in p53-dependent contexts .

• Knockdown verification: Perform siRNA or CRISPR-mediated knockdown of SHISA5 to confirm antibody specificity. The disappearance or reduction of signal in Western blot or immunofluorescence confirms target specificity .

• Epitope blocking: Pre-incubate the antibody with the immunizing peptide before application. The absence of signal confirms specificity to the target epitope .

• Multiple antibody comparison: Use different antibodies targeting distinct SHISA5 epitopes (e.g., comparing results from antibodies targeting AA 75-102 versus AA 56-137) to verify consistent detection patterns .

• Apoptosis induction: Validate functional specificity by inducing apoptosis using DNA-damaging agents that activate p53, then measure SHISA5 upregulation as an indicator of p53 pathway activation .

What experimental controls should be included when using SHISA5 antibodies in apoptosis studies?

When designing experiments to study SHISA5 in apoptotic pathways, include these essential controls:

• Positive apoptosis controls: Treat cells with established apoptosis inducers (staurosporine, UV radiation) alongside your experimental conditions to confirm SHISA5 involvement in the apoptotic response .

• p53 status verification: Include both p53 wild-type and p53-null cells to differentiate between p53-dependent and p53-independent SHISA5 functions .

• Caspase inhibition: Since SHISA5 functions in a caspase-dependent manner, include samples treated with pan-caspase inhibitors (e.g., Z-VAD-FMK) to determine if SHISA5's effects are mediated through caspase activation .

• Subcellular fractionation controls: When examining SHISA5 localization, include markers for specific cellular compartments (e.g., PARP for nucleus, GAPDH for cytosol) .

• Antibody specificity controls: Include primary antibody omission, isotype controls (matching the IgG class of your SHISA5 antibody), and peptide competition controls .

• Time-course experiments: Monitor SHISA5 expression at multiple time points after apoptosis induction to capture the temporal dynamics of its involvement .

How can I optimize SHISA5 antibody-based immunoprecipitation for protein interaction studies?

Optimizing immunoprecipitation (IP) with SHISA5 antibodies requires careful consideration of several parameters:

• Antibody selection: Choose SHISA5 antibodies specifically validated for IP applications, such as the AA 56-137 antibody (ABIN7166112) with recommended dilutions of 1:200-1:2000 .

• Lysate preparation: Use buffers that preserve protein-protein interactions while effectively extracting SHISA5. For membrane-associated proteins like SHISA5, include mild detergents (0.5-1% NP-40 or Triton X-100) in your lysis buffer .

• Cross-linking considerations: For transient or weak interactions, consider using membrane-permeable crosslinkers before cell lysis to stabilize protein complexes involving SHISA5 .

• Pre-clearing strategy: Pre-clear lysates with protein G beads (for rabbit host antibodies) to reduce non-specific binding. The AA 56-137 antibody is Protein G purified, making this step particularly important .

• Antibody incubation conditions: Optimize temperature and duration - typically 4°C overnight provides the best balance between specific binding and minimal degradation .

• Washing stringency gradient: Perform sequential washes with increasing stringency to remove non-specific interactions while preserving specific SHISA5 protein complexes .

• Elution method selection: Choose between denaturing (SDS buffer) or non-denaturing (peptide competition) elution methods depending on downstream applications .

What approaches can resolve contradictory results when using different SHISA5 antibodies?

When faced with contradictory results using different SHISA5 antibodies, implement this systematic troubleshooting approach:

• Epitope mapping analysis: Compare the binding regions of each antibody (AA 75-102, AA 56-137, AA 1-137) to determine if they recognize different SHISA5 isoforms or if post-translational modifications might affect epitope accessibility .

• Isoform-specific detection: SHISA5 has multiple isoforms (evidenced by the various NCBI accession numbers: NP_001258994 through NP_057563) . Different antibodies may preferentially detect specific isoforms, explaining apparent contradictions.

• Antibody validation comparison: Review the validation methods used for each antibody. The AA 75-102 antibody is purified through protein A column followed by peptide affinity purification, while the AA 56-137 antibody undergoes Protein G purification .

• Application-specific optimization: Systematically adjust protocols for each antibody based on their recommended applications. For example, the AA 56-137 antibody is validated for ELISA, IHC, IF, and IP, while the AA 75-102 antibody is specifically validated for WB .

• Combined approach validation: Use multiple detection methods simultaneously (e.g., IF and WB) with the same samples to cross-validate findings across techniques .

• Confirmatory genetic approaches: Implement CRISPR-Cas9 or siRNA knockdown of SHISA5 to definitively determine which antibody most accurately reflects true SHISA5 expression and localization .

What are the optimal protocols for using SHISA5 antibodies in Western blotting?

When using SHISA5 antibodies for Western blotting, follow these protocol considerations for optimal results:

• Sample preparation: For SHISA5 (approximately 25.6 kDa), use RIPA buffer supplemented with protease inhibitors. Since SHISA5 is involved in apoptosis pathways, add phosphatase inhibitors to preserve phosphorylation states .

• Gel selection: Use 12-15% polyacrylamide gels to properly resolve SHISA5's ~25.6 kDa band .

• Transfer conditions: For SHISA5, transfer at 100V for 60 minutes using PVDF membranes, which typically provide better sensitivity for lower abundance proteins like SHISA5 .

• Blocking optimization: Use 5% non-fat dry milk in TBST for 1 hour at room temperature. For phospho-specific detection, substitute with 5% BSA .

• Antibody incubation: For the AA 75-102 antibody, use a 1:1000 dilution in blocking buffer and incubate overnight at 4°C with gentle rocking .

• Washing protocol: Perform 4-5 washes with TBST, 5 minutes each, to minimize background while preserving specific signal .

• Detection system selection: For unconjugated primary antibodies, use an appropriate HRP-conjugated secondary antibody followed by enhanced chemiluminescence detection .

• Expected results interpretation: Anticipate a band at approximately 25.6 kDa for SHISA5, with potential additional bands representing different isoforms or post-translationally modified forms .

How can I effectively use SHISA5 antibodies in immunohistochemistry and immunofluorescence?

For effective use of SHISA5 antibodies in IHC and IF applications, follow these methodological guidelines:

• Fixation method selection: For IHC, 10% neutral buffered formalin fixation is recommended. For IF, 4% paraformaldehyde provides good epitope preservation while maintaining cellular architecture .

• Antigen retrieval optimization: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is generally effective for SHISA5 detection. For challenging samples, try EDTA buffer (pH 9.0) as an alternative .

• Blocking parameters: Block with 10% normal serum from the species of the secondary antibody for 1 hour at room temperature to reduce non-specific binding .

• Primary antibody dilution: For the AA 56-137 antibody, use dilutions of 1:20-1:200 for IHC and 1:50-1:500 for IF applications. Incubate overnight at 4°C in a humidified chamber .

• Signal amplification options: For low-expression samples, consider tyramide signal amplification to enhance detection sensitivity while maintaining specificity .

• Counterstaining selection: For IHC, hematoxylin provides good nuclear contrast. For IF, DAPI or Hoechst can be used for nuclear visualization, particularly useful when studying SHISA5's role in nuclear-initiated apoptotic events .

• Multiplex considerations: When performing multiplex IF with other apoptotic markers, select antibodies raised in different host species to avoid cross-reactivity. The rabbit polyclonal nature of many SHISA5 antibodies allows combination with mouse monoclonal antibodies targeting other proteins .

• Controls implementation: Include tissue sections known to express SHISA5 (positive control) and primary antibody omission controls (negative control) .

What strategies can maximize reproducibility when using SHISA5 antibodies across different experimental batches?

To ensure consistent results across experimental batches when using SHISA5 antibodies, implement these reproducibility strategies:

• Antibody validation standardization: Perform and document comprehensive validation for each new lot of SHISA5 antibody using Western blot against a consistent positive control lysate .

• Aliquoting practice: Upon receipt, aliquot antibodies into single-use volumes to avoid repeated freeze-thaw cycles. For the AA 75-102 antibody, store at -20°C for long-term or 2-8°C for up to 6 months .

• Standard curve inclusion: For quantitative applications, include a standard curve in each experiment using recombinant SHISA5 protein at known concentrations .

• Reference sample utilization: Maintain a laboratory reference sample with established SHISA5 expression levels to normalize across experiments .

• Protocol documentation precision: Maintain detailed protocols including specific antibody dilutions, incubation times and temperatures, and lot numbers .

• Imaging parameters standardization: For fluorescence applications, establish and record fixed exposure settings, gain, and offset values for SHISA5 detection .

• Quantification method consistency: Use consistent image analysis and quantification methods across experiments, ideally with automated scripts to reduce operator variability .

• Environmental variable control: Monitor and record laboratory temperature and humidity, as these can affect antibody binding kinetics and background levels .

How can I design experiments to determine the subcellular localization of SHISA5 using available antibodies?

To accurately determine SHISA5 subcellular localization, design experiments incorporating these methodological considerations:

• Antibody selection for localization studies: Choose SHISA5 antibodies specifically validated for immunofluorescence, such as the AA 56-137 antibody (ABIN7166112) or the FITC-conjugated version for direct detection .

• Subcellular marker co-localization panel: Co-stain with established markers for different cellular compartments:

  • ER: Calnexin or PDI

  • Golgi: GM130

  • Nucleus: Lamin B1

  • Mitochondria: TOM20

  • Plasma membrane: Na+/K+ ATPase

• Fixation method comparison: Compare multiple fixation methods (paraformaldehyde, methanol, acetone) as they can differentially preserve epitopes and cellular structures .

• Confocal microscopy implementation: Use confocal microscopy with z-stack acquisition to precisely determine the three-dimensional localization of SHISA5 within cellular compartments .

• Super-resolution microscopy application: For detailed co-localization studies, employ super-resolution techniques (STED, PALM, or STORM) to resolve structures beyond the diffraction limit .

• Live-cell imaging considerations: For dynamic localization studies, consider using cells transfected with SHISA5-GFP fusion proteins alongside immunostaining of endogenous SHISA5 for validation .

• Subcellular fractionation correlation: Complement imaging data with biochemical subcellular fractionation followed by Western blotting using the AA 75-102 antibody (ABIN1538603) to confirm localization patterns .

• Stimulus-dependent translocation assessment: Evaluate changes in SHISA5 localization upon apoptosis induction with DNA-damaging agents or other p53-activating stimuli .

How can SHISA5 antibodies be used to investigate the relationship between SHISA5 and p53-dependent apoptosis?

To investigate SHISA5's role in p53-dependent apoptosis, implement these methodological approaches:

• Co-immunoprecipitation strategy: Use SHISA5 antibodies suitable for IP (AA 56-137) to pull down protein complexes and probe for p53 and other apoptotic pathway components .

• Proximity ligation assay implementation: Combine SHISA5 antibodies with p53 antibodies in proximity ligation assays to visualize and quantify direct interactions in situ .

• Cell line panel analysis: Compare SHISA5 expression and localization in isogenic cell lines with different p53 status (wild-type, null, mutant) following DNA damage or other apoptotic stimuli .

• Chemical inhibitor matrix: Combine SHISA5 detection with chemical inhibitors of different apoptotic pathway components (caspase inhibitors, p53 inhibitors, MDM2 inhibitors) to dissect the pathway hierarchy .

• Time-course evaluation: Monitor SHISA5 expression and localization at multiple time points after p53 activation to establish temporal relationships in the apoptotic cascade .

• Chromatin immunoprecipitation sequential analysis: If investigating transcriptional regulation, perform ChIP with p53 antibodies followed by qPCR for the SHISA5 promoter region to establish direct transcriptional regulation .

• CRISPR screen integration: Combine SHISA5 antibody-based detection with CRISPR screens targeting apoptotic pathway components to identify synthetic interactions .

What advanced imaging techniques can be combined with SHISA5 antibodies for in-depth cellular analysis?

Combining SHISA5 antibodies with advanced imaging techniques can provide deeper insights into its function:

• Stimulated emission depletion (STED) microscopy application: Use SHISA5 antibodies conjugated with STED-compatible fluorophores to achieve super-resolution imaging of SHISA5 localization relative to apoptotic machinery .

• Fluorescence resonance energy transfer (FRET) implementation: Combine SHISA5 antibodies with antibodies against potential interaction partners labeled with FRET-compatible fluorophores to detect protein-protein interactions in situ .

• Fluorescence recovery after photobleaching (FRAP) integration: For live-cell studies, use fluorescently tagged SHISA5 to assess protein dynamics and mobility in different cellular compartments during apoptosis .

• Correlative light-electron microscopy (CLEM) application: Combine immunofluorescence detection of SHISA5 with electron microscopy to correlate its localization with ultrastructural features of apoptosis .

• Expansion microscopy adaptation: Apply physical tissue expansion techniques with SHISA5 immunostaining to achieve super-resolution-like imaging on conventional microscopes .

• Light-sheet microscopy utilization: For 3D tissue samples or organoids, use light-sheet microscopy with SHISA5 antibodies to visualize apoptotic patterns across large volumes with minimal photobleaching .

• Multi-epitope ligand cartography (MELC) implementation: Use iterative staining and bleaching cycles to visualize SHISA5 alongside dozens of other markers in the same sample to create comprehensive protein maps .

How can SHISA5 antibodies be used in high-throughput screening applications?

To adapt SHISA5 antibodies for high-throughput screening applications, consider these methodological strategies:

• Automated immunofluorescence optimization: Optimize SHISA5 antibody staining protocols (concentrations, incubation times) for compatibility with automated liquid handling systems and high-content imaging platforms .

• Multiplex assay development: Combine SHISA5 antibodies with other apoptotic markers in multiplexed assays to simultaneously assess multiple pathway components. Select antibodies raised in different host species or use directly conjugated antibodies to avoid cross-reactivity .

• ELISA-based screening adaptation: Develop ELISA assays using SHISA5 antibodies (especially those validated for ELISA applications like AA 56-137 and AA 75-102) to quantify SHISA5 levels across large sample sets .

• Reverse phase protein array implementation: Immobilize cell/tissue lysates on nitrocellulose-coated slides and probe with SHISA5 antibodies to rapidly analyze hundreds of samples simultaneously .

• Flow cytometry protocol development: Optimize intracellular staining protocols using SHISA5 antibodies for flow cytometry to analyze large cell populations and potentially sort cells based on SHISA5 expression levels .

• Bead-based multiplex assay creation: Develop Luminex or similar bead-based assays using SHISA5 antibodies for simultaneous quantification of multiple proteins in apoptotic pathways .

• Reporter system correlation: Validate high-throughput compatible reporter systems (luciferase, fluorescent proteins) that correlate with SHISA5 expression or activity, using SHISA5 antibodies as the validation tool .