stx18 Antibody

What is STX18 and why is it important in cellular biology?

STX18 (Syntaxin 18) is an ER-resident SNARE protein that plays a critical role in retrograde vesicular transport between the Golgi apparatus and endoplasmic reticulum. Beyond its role in membrane trafficking, STX18 has been identified as a substrate for the intramembrane protease SPP and has emerging functions in DNA damage response pathways. It is relatively long-lived compared to many cellular proteins and has been implicated in cancer cell radioresistance, making it an important target for both basic cell biology and translational cancer research .

Which STX18 antibody formats are available for research applications?

STX18 antibodies are available in multiple formats including monoclonal mouse antibodies (such as clone 2E5) and polyclonal rabbit antibodies. The monoclonal antibodies are available in specialized formats such as azide-free and BSA-free preparations for sensitive applications. These antibodies have been validated across multiple species including human, mouse, and rat samples. Researchers should select the appropriate format based on their experimental needs, with monoclonals offering higher specificity and polyclonals potentially providing greater sensitivity .

What are the validated applications for STX18 antibodies?

STX18 antibodies have been validated for multiple applications including:

• Western blotting (WB)

• Enzyme-linked immunosorbent assay (ELISA)

• Immunohistochemistry (IHC)

• Immunohistochemistry on paraffin-embedded tissues (IHC-P)

• Immunocytochemistry/Immunofluorescence (ICC/IF)

• Sandwich ELISA

The recommended dilutions vary by application, with IHC typically using 1:500-1:1000 dilutions and ICC/IF requiring 1-4 μg/ml. For optimal results, researchers should experimentally determine the ideal concentration for their specific experimental conditions and sample types .

What are the best practices for using STX18 antibodies in Western blot applications?

For optimal Western blot results with STX18 antibodies:

• Sample preparation: Prepare cell lysates in RIPA or NP-40 buffer containing protease inhibitors to prevent degradation

• Protein loading: Load 20-40 μg of total protein per lane

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal separation

• Transfer conditions: Transfer to PVDF membrane at 100V for 1 hour or 30V overnight

• Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody: Incubate with STX18 antibody at experimentally determined dilution (typically starting at 1:1000) overnight at 4°C

• Detection controls: Include positive controls such as PC-12 or Raw 264.7 cell lysates which have been validated for STX18 detection

• Expected results: Anticipate a band at approximately 42 kDa for full-length STX18, with potential cleavage fragments at lower molecular weights when studying proteolytic processing

The antibody has been validated to detect STX18 expression in cell lines such as PC-12 and Raw 264.7, making these useful positive controls .

How should STX18 antibodies be stored and handled to maintain activity?

To maintain optimal antibody performance:

• Upon receipt, immediately aliquot the antibody to minimize freeze-thaw cycles

• Store at -20°C or -80°C in small working volumes (typically 10-20 μl)

• Avoid more than 2-3 freeze-thaw cycles as this can lead to antibody denaturation and loss of activity

• When thawing, place on ice and use immediately or return to storage

• For diluted working solutions, store at 4°C and use within 1-2 weeks

• If using BSA-free and azide-free formulations, consider adding a protein carrier (0.1-1% BSA) for longer-term storage of diluted antibody

• Always centrifuge briefly before opening the antibody vial to collect all liquid at the bottom

Following these guidelines will help maintain antibody specificity and sensitivity throughout your research project timeline .

What controls should be included when using STX18 antibodies in immunohistochemistry?

When performing immunohistochemistry with STX18 antibodies, the following controls are essential:

• Positive tissue control: Human salivary gland has been validated for STX18 expression and serves as an excellent positive control

• Negative controls:

  • Omission of primary antibody (secondary antibody only)

  • Isotype control (matching IgG at the same concentration)

  • Tissue known to lack STX18 expression

• Antigen competition: Pre-incubation of antibody with immunizing peptide to confirm specificity

• Concentration gradient: Testing multiple antibody concentrations (typically 1-5 μg/ml)

• Signal validation: Using multiple antibodies targeting different epitopes of STX18

• Knockdown validation: When possible, compare staining between wild-type and STX18-knockdown samples

Recommended antibody concentration is 3 μg/ml for paraffin-embedded tissues, but optimal concentration should be determined experimentally for each tissue type and fixation method .

How can STX18 antibodies be used to study its role in the DNA damage response pathway?

To investigate STX18's role in DNA damage response:

• Radiation-induced damage model:

  • Treat cells with X-radiation (2-10 Gy)

  • Use STX18 antibodies in Western blot and immunofluorescence to monitor changes in expression levels and subcellular localization

  • Compare STX18 levels in radioresistant versus radiosensitive cell lines

• STX18 knockdown experiments:

  • Generate STX18 knockdown cell lines using siRNA or CRISPR-Cas9

  • Assess DNA damage response by measuring γH2AX foci formation

  • Evaluate cell cycle checkpoints via flow cytometry

  • Analyze clonogenic survival following radiation treatment

  • Use STX18 antibodies to confirm knockdown efficiency

• Co-immunoprecipitation:

  • Use STX18 antibodies to pull down protein complexes

  • Analyze interactions with DNA repair proteins

  • Compare protein interactions before and after DNA damage induction

These approaches can reveal how STX18 integrates membrane trafficking with DNA damage response pathways, particularly in the context of cancer radioresistance .

What strategies can resolve non-specific binding issues with STX18 antibodies?

When experiencing non-specific binding with STX18 antibodies:

• Increase blocking stringency:

  • Use 5% BSA instead of milk for blocking

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Consider adding 5% normal serum from the secondary antibody host species

• Antibody optimization:

  • Titrate primary antibody concentration

  • Reduce incubation time or increase washing steps

  • Test different antibody clones targeting different epitopes

• Sample preparation adjustments:

  • Optimize fixation protocols (reduce fixation time)

  • Try antigen retrieval methods (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • For Western blot, increase washing time and detergent concentration

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Reduce secondary antibody concentration

  • Consider using F(ab')2 fragments to reduce Fc receptor binding

• Validation strategies:

  • Verify specificity with peptide competition assays

  • Include STX18 knockdown samples as negative controls

These approaches can help distinguish specific STX18 signal from background, particularly in complex tissue samples .

How can STX18 proteolytic processing be studied using antibodies?

To investigate STX18 processing by the intramembrane protease SPP:

• Experimental design:

  • Treat cells with proteasome inhibitors (e.g., epoxomicin) to stabilize cleaved fragments

  • Use cycloheximide chase experiments to assess protein turnover rates

  • Apply SPP inhibitors such as (Z-LL)2-ketone to block cleavage

• Antibody-based detection strategies:

  • Use antibodies recognizing different domains of STX18 to track specific fragments

  • Perform Western blotting with gradient gels (8-16%) to resolve both full-length and cleaved products

  • Combine with subcellular fractionation to detect cytosolic accumulation of cleaved fragments

• Quantification approach:

  • Measure the ratio of full-length to cleaved STX18

  • Track degradation kinetics by time-course experiments

  • Compare processing efficiency under different cellular conditions

• Controls:

  • Co-express wild-type SPP versus catalytically inactive SPP-DA mutant

  • Include other SNARE proteins (e.g., STX5) as specificity controls

  • Use cells with varying levels of endogenous SPP expression

This methodology has revealed that STX18 is a bona fide SPP substrate, with cleaved fragments being released into the cytosol and subsequently degraded by the proteasome .

How does STX18 contribute to cancer cell radioresistance, and how can antibodies help study this mechanism?

STX18's role in cancer radioresistance can be investigated using antibodies through:

• Expression analysis in cancer models:

  • Compare STX18 protein levels across radioresistant and radiosensitive cell lines

  • Assess STX18 expression in patient-derived xenografts before and after radiation treatment

  • Correlate STX18 expression with clinical radiotherapy outcomes in patient samples

• Mechanistic investigations:

  • Use immunofluorescence to track STX18 relocalization following radiation

  • Employ co-immunoprecipitation with STX18 antibodies to identify radiation-induced protein interactions

  • Study post-translational modifications of STX18 after radiation exposure

• Functional analyses:

  • Monitor STX18-dependent gene expression programs using antibodies for ChIP-seq

  • Assess impact on DNA damage repair by measuring γH2AX foci clearance

  • Analyze effects on cell cycle checkpoints and mitotic catastrophe

Research has shown that STX18 downregulation impairs DNA damage-induced cell cycle checkpoints and leads to cell death by mitotic catastrophe. Additionally, STX18 regulates epithelial-mesenchymal transition markers and affects migration and invasion capacity, suggesting it controls both primary tumor growth and metastatic potential following radiotherapy .

What role does STX18-AS1 play in cardiac development, and how can this be studied?

STX18-AS1 is a long non-coding RNA implicated in atrial septal defects. To study its role:

• Expression analysis:

  • Use RT-qPCR to measure STX18-AS1 expression during cardiac development

  • Perform in situ hybridization to localize STX18-AS1 in developing heart tissue

  • Compare expression between normal and diseased cardiac samples

• Functional studies:

  • Generate STX18-AS1 knockdown models using CRISPR techniques

  • Assess impact on cardiomyocyte differentiation from human embryonic stem cells

  • Monitor cardiac-specific gene expression, particularly NKX2-5

• Molecular mechanism investigation:

  • Perform Chromatin Isolation by RNA Purification (ChIRP) to study STX18-AS1 interaction with chromatin

  • Use ChIP-qPCR to measure histone modifications (H3K4me3, H3K27me3) at cardiac gene promoters

  • Analyze interaction with chromatin remodeling complexes like SET1/MLL complex

Research has demonstrated that STX18-AS1 knockdown reduces H3K4me3 around the NKX2-5 gene by approximately 70%, corresponding with reduced NKX2-5 expression. STX18-AS1 interacts with WDR5, a scaffold protein of the SET1/MLL complex necessary for H3K4 trimethylation, suggesting it regulates cardiac development through epigenetic mechanisms .

How can STX18 antibodies be applied in studying neurodegenerative diseases?

Although the provided search results don't directly address STX18 in neurodegeneration, its role as an ER-resident SNARE protein suggests potential applications:

• ER stress investigations:

  • Use STX18 antibodies to monitor protein levels during ER stress conditions

  • Assess STX18 distribution in neurons exposed to neurotoxic proteins

  • Compare STX18 processing in control versus disease models

• Vesicular trafficking studies:

  • Employ co-localization analysis with STX18 antibodies and markers of ER-Golgi intermediate compartment

  • Track anterograde/retrograde transport defects in neuronal models

  • Measure interaction with other SNARE components under disease conditions

• Proteostasis analysis:

  • Monitor STX18 degradation kinetics in presence of misfolded proteins

  • Assess SPP-mediated processing of STX18 in neurodegenerative models

  • Investigate potential sequestration of STX18 in protein aggregates

• Therapeutic target validation:

  • Use STX18 antibodies to confirm target engagement of compounds affecting ER-Golgi trafficking

  • Monitor STX18 levels following treatment with proteostasis modulators

  • Validate STX18 pathway interventions in disease models

These approaches could reveal novel roles for STX18 in neuronal health and disease, potentially identifying new therapeutic targets for neurodegenerative conditions.

What protocols are recommended for using STX18 antibodies in multiplexed imaging studies?

For multiplexed imaging with STX18 antibodies:

• Antibody selection and validation:

  • Choose STX18 antibodies raised in different host species than other target antibodies

  • Validate antibody specificity with appropriate controls

  • Test for cross-reactivity with other primary and secondary antibodies in the panel

• Sample preparation:

  • Optimize fixation protocols (4% paraformaldehyde for 10-15 minutes)

  • Use sequential antigen retrieval if necessary

  • Apply appropriate blocking (5% normal serum from secondary antibody host species)

• Staining strategies:

  • Sequential staining: Complete one staining cycle before starting the next

  • Simultaneous staining: Apply compatible primary antibodies together

  • Consider using directly conjugated STX18 antibodies to eliminate secondary antibody issues

• Signal separation:

  • Select fluorophores with minimal spectral overlap

  • Apply spectral unmixing algorithms for closely overlapping signals

  • Include single-stained controls for each fluorophore

• Image acquisition:

  • Use sequential scanning for confocal microscopy

  • Apply appropriate exposure settings to avoid bleed-through

  • Acquire z-stacks to capture the full subcellular distribution

When co-staining with markers of vesicular compartments, STX18 antibodies work effectively at concentrations of 1-4 μg/ml for immunofluorescence applications .

How can quantitative analysis of STX18 expression be performed across different tissue samples?

For quantitative analysis of STX18 expression:

• Sample standardization:

  • Use consistent tissue processing protocols

  • Prepare tissue microarrays for high-throughput analysis

  • Process all samples simultaneously when possible

• Staining optimization:

  • Determine linear range of STX18 antibody staining

  • Include calibration controls with known STX18 expression levels

  • Apply automated staining platforms for consistency

• Image acquisition standardization:

  • Use identical microscope settings across all samples

  • Acquire images at the same exposure and gain settings

  • Include fluorescence calibration beads or slides

• Quantification methods:

  • Define consistent regions of interest (ROIs)

  • Apply automated analysis algorithms for unbiased quantification

  • Measure both intensity and distribution parameters

• Data normalization:

  • Normalize to housekeeping proteins

  • Apply tissue-specific normalization factors

  • Use z-score transformations for cross-tissue comparisons

• Statistical analysis:

  • Apply appropriate statistical tests for comparison across groups

  • Consider hierarchical clustering for pattern recognition

  • Use machine learning approaches for complex dataset analysis

This approach has been used successfully to compare STX18 expression in normal versus cancerous tissues, revealing its potential role in cancer progression .

What are the best approaches for studying STX18 interactions with other proteins?

To investigate STX18 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use STX18 antibodies conjugated to agarose or magnetic beads

  • Apply mild lysis conditions to preserve protein-protein interactions

  • Include appropriate controls: IgG control, input samples, and reciprocal IP

  • Consider crosslinking to stabilize transient interactions

• Proximity ligation assay (PLA):

  • Combine STX18 antibody with antibodies against potential interacting partners

  • Optimize antibody concentrations and incubation conditions

  • Include appropriate controls: single antibody controls and known interacting pairs

• FRET/BRET analysis:

  • Generate fluorescent protein-tagged STX18 constructs

  • Combine with tagged potential interacting partners

  • Measure energy transfer in live cells

  • Validate findings with co-IP of endogenous proteins

• Mass spectrometry approaches:

  • Perform immunoprecipitation with STX18 antibodies

  • Analyze precipitated complexes by LC-MS/MS

  • Filter against appropriate control IPs

  • Validate top hits by orthogonal methods

Research has demonstrated that STX18 physically interacts with the intramembrane protease SPP, with the catalytically inactive SPP-DA mutant showing stronger interaction due to its substrate-trapping properties. This approach revealed that approximately 10% of total STX18 interacts with SPP-DA, providing insight into the kinetics of this enzyme-substrate relationship .

How might STX18 antibodies be utilized in studying cellular stress responses beyond DNA damage?

STX18 antibodies can be applied to investigate various cellular stress responses:

• ER stress and unfolded protein response:

  • Monitor STX18 expression and localization during ER stress

  • Assess correlation with UPR markers (BiP, CHOP, XBP1s)

  • Track changes in STX18 processing under ER stress conditions

• Oxidative stress:

  • Evaluate STX18 distribution following oxidative damage

  • Measure post-translational modifications (oxidation, nitrosylation)

  • Assess impact on membrane trafficking during redox imbalance

• Nutrient deprivation and autophagy:

  • Track STX18 during starvation-induced autophagy

  • Investigate potential roles in autophagosome formation

  • Study interaction with autophagy machinery components

• Hypoxic stress:

  • Monitor STX18 expression under hypoxic conditions

  • Assess correlation with HIF-1α stabilization

  • Evaluate changes in vesicular trafficking during hypoxia

These applications could reveal how membrane trafficking systems integrate with cellular stress response pathways, potentially identifying novel therapeutic targets for stress-related pathologies .

What methodological advances might improve STX18 detection in challenging samples?

Several advanced techniques can enhance STX18 detection in difficult samples:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) to enhance fluorescence detection

  • Rolling circle amplification for ultrasensitive detection

  • Quantum dot-conjugated secondary antibodies for improved signal-to-noise ratio

• Sample preparation enhancements:

  • Optimized antigen retrieval methods for formalin-fixed tissues

  • Tissue clearing techniques for thick section imaging

  • Hydrogel embedding for structure preservation

• Advanced microscopy approaches:

  • Super-resolution microscopy (STORM, PALM, STED) for nanoscale localization

  • Expansion microscopy for physical magnification of structures

  • Light sheet microscopy for rapid 3D imaging with reduced photobleaching

• Combinatorial detection strategies:

  • Antibody-guided CRISPR-Cas9 systems for highly specific detection

  • Proximity-dependent biotin identification (BioID) for interaction mapping

  • Antibody-DNA conjugates for digital quantification (Immuno-SABER)

These methodological advances could enable more sensitive and specific detection of STX18 in complex samples such as patient tissues, organoids, or single cells .

How might the role of STX18 in epithelial-mesenchymal transition inform cancer therapy approaches?

STX18's role in epithelial-mesenchymal transition (EMT) suggests several therapeutic implications:

• Radiosensitization strategies:

  • Develop STX18-targeted approaches to enhance radiotherapy efficacy

  • Combine STX18 inhibition with DNA damage response inhibitors

  • Monitor EMT markers as biomarkers of treatment response

• Metastasis prevention:

  • Target STX18-dependent pathways to reduce migration and invasion

  • Develop combination approaches targeting both primary and metastatic disease

  • Identify patient subgroups likely to benefit from STX18-targeted therapies

• Resistance mechanisms:

  • Investigate STX18's role in treatment-induced EMT

  • Study adaptive responses to STX18 inhibition

  • Develop strategies to overcome potential resistance mechanisms

• Biomarker development:

  • Use STX18 antibodies to stratify patients for treatment selection

  • Monitor STX18 levels during treatment as response indicators

  • Develop multiplexed assays including STX18 and EMT markers