ST8SIA5 Antibody

What is ST8SIA5 and what are its primary biological functions?

ST8SIA5 is a type II membrane protein typically located in the Golgi apparatus and functions as a member of glycosyltransferase family 29. It plays a critical role in ganglioside synthesis, specifically catalyzing the formation of GD1c, GT1a, GQ1b, GP1c, and GT3 from GD1a, GT1b, GM1b, and GD3 respectively . ST8SIA5 operates alongside proteins such as NCAM1 and ST8SIA2, impacting cell adhesion and signaling processes that are important in neural development and synaptic functions . Its sialyltransferase activity is essential for proper glycosylation patterns in various tissues, particularly in the nervous system.

What alternative names and designations exist for ST8SIA5?

Researchers should be aware of the following alternative designations when searching literature:

• SIAT8E (previous HGNC symbol)

• ST8SiaV

• SIAT8-E

• Alpha-2,8-sialyltransferase 8E

• Sialyltransferase 8E (alpha-2,8-polysialytransferase)

• Sialyltransferase 8 (alpha-2,8-sialyltransferase) E

Understanding these alternative designations is crucial when conducting comprehensive literature reviews and database searches for ST8SIA5-related research.

What applications are validated for commercially available ST8SIA5 antibodies?

ST8SIA5 antibodies have been validated for multiple experimental applications, primarily:

• Immunohistochemistry (IHC-P)

• Immunocytochemistry/Immunofluorescence (ICC/IF)

• Western Blotting (WB)

• Enzyme-linked Immunosorbent Assay (ELISA)

For example, the antibody ab184777 has been specifically validated for IHC-P and ICC/IF with human samples . When selecting an antibody, researchers should verify which applications have been directly tested by manufacturers versus applications that are predicted to work based on homology.

How should I evaluate the specificity of different ST8SIA5 antibodies?

Evaluate antibody specificity by:

• Reviewing validation data provided by manufacturers

• Examining immunohistochemical images showing expected cellular localization patterns

• Checking for positive cytoplasmic staining in known ST8SIA5-expressing tissues such as:

  • Neurons in cerebral cortex

  • Seminiferous ducts in testicular tissue

  • Plasma membrane and midbody in certain cell lines (e.g., RH-30)

• Confirming the antibody targets the correct protein region - for instance, ab184777 targets a recombinant fragment within Human ST8SIA5 aa 300 to C-terminus

Additionally, consider performing your own validation using positive and negative control tissues or knockout/knockdown models where available.

What are the optimal conditions for immunohistochemical detection of ST8SIA5?

For optimal IHC detection of ST8SIA5:

• Fixation and Embedding: Standard formalin fixation and paraffin embedding protocols are suitable for ST8SIA5 detection

• Antigen Retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective

• Antibody Concentration: Start with manufacturer's recommended dilution (e.g., 2 μg/ml for ab184777 in ICC/IF applications)

• Detection Systems: Both chromogenic (DAB) and fluorescent secondary detection systems have been successfully used

• Expected Pattern: Look for cytoplasmic staining in neurons (cerebral cortex) and seminiferous ducts (testis)

• Controls: Include human cerebral cortex as a positive control tissue due to reliable ST8SIA5 expression

Special attention should be paid to membrane protein preservation during tissue processing, as improper fixation can disrupt the Golgi localization pattern of ST8SIA5.

What are the key considerations for using ST8SIA5 antibodies in co-localization studies?

When performing co-localization studies with ST8SIA5:

• Subcellular Markers: Include Golgi apparatus markers (GM130, TGN46) to confirm ST8SIA5's expected localization

• Other Pathway Proteins: Consider co-staining with related sialyltransferases (ST8SIA1-4) or ganglioside synthases

• Sequential Staining: If using multiple primary antibodies from the same species, employ sequential staining protocols with careful blocking steps

• Spectral Separation: Ensure fluorophores have sufficient spectral separation to avoid bleed-through

• Image Acquisition: Use confocal microscopy with appropriate controls for quantitative co-localization analysis

• Resolution Considerations: The compact nature of the Golgi apparatus requires high-resolution imaging

When co-localizing ST8SIA5 with NCAM1 or ST8SIA2, optimize antibody concentrations to account for potentially different expression levels of these interacting proteins .

How can I troubleshoot weak or absent signal when using ST8SIA5 antibodies?

If experiencing weak or absent signal:

• Antibody Concentration: Titrate antibody concentration; ST8SIA5 may require higher concentrations than typical membrane proteins

• Antigen Retrieval: Extend antigen retrieval time or try alternative buffers (EDTA buffer pH 9.0 vs. citrate buffer pH 6.0)

• Detection System: Switch to more sensitive detection systems (e.g., tyramide signal amplification)

• Sample Processing: Ensure samples were properly fixed and processed to preserve Golgi structure

• Expression Levels: Verify ST8SIA5 expression in your tissue/cell type of interest - expression may be tissue-specific

• Antibody Storage: Check antibody storage conditions; improper storage can reduce activity

• Epitope Accessibility: Consider mild detergent permeabilization to improve access to Golgi-resident proteins

When specifically troubleshooting immunofluorescence, photobleaching can be reduced by adding anti-fade reagents and minimizing exposure to light during processing.

What could cause non-specific or unexpected staining patterns with ST8SIA5 antibodies?

For non-specific or unexpected staining:

• Cross-Reactivity: Check for potential cross-reactivity with other sialyltransferase family members (particularly ST8SIA1-4, ST8SIA6)

• Blocking: Increase blocking time/concentration to reduce non-specific binding

• Secondary Antibody: Test secondary antibody alone to rule out non-specific binding

• Tissue Autofluorescence: For IF applications, use appropriate quenching methods for tissue autofluorescence

• Specificity Validation: Perform peptide competition assays to confirm antibody specificity

• Post-translational Modifications: Consider that unexpected patterns might represent previously undocumented post-translational modifications

• Splice Variants: Check if the antibody epitope is present in all known splice variants of ST8SIA5

If unexpected nuclear staining occurs, this may indicate non-specific binding, as ST8SIA5 is typically localized to the Golgi apparatus.

How can ST8SIA5 antibodies be utilized to study ganglioside synthesis pathways?

To study ganglioside synthesis pathways:

• Enzyme Activity Correlation: Correlate ST8SIA5 protein expression (by immunostaining) with sialyltransferase activity assays

• Pathway Mapping: Use ST8SIA5 antibodies alongside antibodies against:

  • Upstream enzymes: GM3 synthase, GD3 synthase

  • Downstream products: antibodies specific to gangliosides GD1c, GT1a, GQ1b

• Knockdown Studies: Combine with siRNA knockdown of ST8SIA5 to observe effects on ganglioside profiles

• Time-course Experiments: Track ST8SIA5 expression during developmental stages or disease progression

• Co-immunoprecipitation: Use ST8SIA5 antibodies for co-IP to identify interaction partners in the ganglioside synthesis pathway

This approach allows for comprehensive mapping of the entire ganglioside synthesis pathway and identification of rate-limiting steps.

What considerations are important when studying ST8SIA5 in neurological disease models?

When investigating ST8SIA5 in neurological disease contexts:

• Expression Pattern Changes: Compare ST8SIA5 expression patterns between healthy and diseased neural tissues

• Ganglioside Profile Analysis: Correlate ST8SIA5 expression with changes in complex ganglioside profiles

• Neuronal Subtype Specificity: Use dual-labeling with neuronal subtype markers to identify cell type-specific expression changes

• Regional Variations: Account for brain region-specific differences in ST8SIA5 expression

• Developmental Timing: Consider developmental timing of ST8SIA5 expression in relation to disease onset

• Signaling Pathways: Investigate interactions between ST8SIA5 and neuronal signaling pathways

The interaction between ST8SIA5, NCAM1, and ST8SIA2 may be particularly relevant in neurodevelopmental disorders, as these proteins impact cell adhesion and signaling processes important in neural development and synaptic functions .

What are the recommended storage and handling conditions for ST8SIA5 antibodies?

For optimal preservation of ST8SIA5 antibodies:

• Storage Temperature: Store at -20°C for long-term storage or at 4°C for short-term storage

• Aliquoting: Prepare small aliquots to avoid repeated freeze-thaw cycles

• Buffer Considerations: Store in manufacturer's recommended buffer, typically with protein stabilizers

• Working Dilutions: Prepare fresh working dilutions on the day of use

• Centrifugation: Briefly centrifuge antibody vials before opening to collect liquid at the bottom

• Contamination Prevention: Use sterile techniques to prevent microbial contamination

• Transport: Transport on ice or with cold packs

Antibody degradation can be monitored by including positive controls in each experiment and comparing signal intensity over time.

What species reactivity is available for commercial ST8SIA5 antibodies?

Commercial ST8SIA5 antibodies are available with reactivity against:

• Human: Multiple validated antibodies, including:

  • ABIN7143803 (validated for ELISA, IHC, WB)

  • ABIN7143806 (validated for ELISA)

  • ABIN7143804 (validated for ELISA)

  • ab184777 (validated for IHC-P, ICC/IF)

• Mouse: Limited options, including:

  • ABIN7556663 (mouse ST8SIA5 protein from HEK-293 cells)

• Other Species: Likely to work in species with high sequence homology, though specific validation may be lacking

When selecting antibodies for cross-species applications, researchers should consider sequence homology in the epitope region and whether the antibody has been specifically validated in the species of interest.