spx Antibody

What is SPX/Spexin and why is it significant in research?

Spexin (SPX), also known as Chromosome 12 Open Reading Frame 39 (C12orf39), MGC10946, or NPQ, is a peptide hormone belonging to the spexin family. It is a 13.302 kDa protein encoded by the SPX gene located on chromosome 12p12.1 in humans . SPX has been identified as a peptide hormone through evolutionary sequence modeling and hidden Markov model screening of the human proteome .

Research significance stems from SPX's inhibitory effect on adrenocortical cell proliferation with minor stimulation of corticosteroid release. This bioactivity suggests potential roles in endocrine regulation, making SPX antibodies essential tools for investigating its expression and function in normal physiology and disease states .

In which tissues is SPX predominantly expressed?

SPX exhibits specific tissue distribution patterns that researchers should consider when designing experiments with SPX antibodies. Expression analysis reveals:

SPX is also secreted via the classical ER/Golgi-dependent pathway into the extracellular medium primarily as a full-length protein without the signal peptide, rather than as a hydrolyzed and amidated peptide . It can be detected extracellularly surrounding villous trophoblastic cells and in serum, which should be considered when designing experiments targeting different cellular compartments .

What validation strategies confirm SPX antibody specificity?

Validation of SPX antibody specificity is crucial before conducting experiments. Recommended methodological approaches include:

• Testing on tissues known to express SPX positively (pancreas, testis, kidney) versus negative control tissues

• Western blot analysis demonstrating the expected 13.3 kDa band

• Blocking peptide competition assays to confirm binding specificity

• Immunoprecipitation followed by mass spectrometry analysis

• Comparison of staining patterns across multiple validated antibodies targeting different epitopes

For research publication, documenting these validation steps enhances experimental rigor. Commercial providers often validate antibody specificity on known positive and negative tissues, and reviewing these validation images should be part of antibody selection process .

How should researchers design immunodetection experiments for SPX?

When designing experiments to detect SPX using antibodies, researchers should consider its secretory nature and cellular localization. SPX is secreted via the ER/Golgi-dependent pathway and can be found in cytoplasmic vesicles, secretory vesicles, extracellular space, and serum . Experimental designs should account for:

• Sample preparation methods appropriate for secreted proteins:

  • For tissue sections: proper fixation to preserve secretory vesicles

  • For cell culture: collection of both cellular fraction and conditioned media

  • For serum: standardized collection and processing protocols

• Detection methods based on localization:

  • Immunohistochemistry: focus on cytoplasmic vesicles and extracellular spaces

  • Immunofluorescence: co-staining with secretory vesicle markers

  • ELISA: optimization for serum or media samples

• Time course considerations:

  • Secretion kinetics may influence detection sensitivity

  • Sampling at multiple timepoints to capture dynamic secretion patterns

Statistical power calculations should determine appropriate sample sizes, with triplicate technical replicates as minimum standard for quantitative assessments .

What are the optimal protocols for SPX antibody application in immunohistochemistry?

For optimal immunohistochemistry results with SPX antibodies, the following methodological approach is recommended:

• Tissue preparation:

  • Fresh tissues should be fixed in 4% paraformaldehyde (12-24 hours)

  • Paraffin-embedded sections cut at 4-6 μm thickness

  • Antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes

• Antibody application:

  • Blocking with 5% normal serum from the species of secondary antibody origin

  • Primary SPX antibody dilution: typically 1:200-1:500 (optimize for each antibody)

  • Incubation overnight at 4°C in humidity chamber

  • Secondary antibody application for 1-2 hours at room temperature

• Detection and controls:

  • Include pancreas or testis as positive control tissues

  • Include isotype control antibody at matching concentration

  • Use chromogenic detection (DAB) for localization studies

  • Use fluorescent detection for co-localization with vesicle markers

• Counterstaining:

  • Light hematoxylin for chromogenic detection

  • DAPI for nuclear visualization in fluorescent detection

Optimization may be required for each tissue type and antibody source, with titration experiments to determine optimal concentration .

What approaches can resolve cross-reactivity issues with SPX antibodies?

When encountering potential cross-reactivity with SPX antibodies, methodological approaches to resolve these issues include:

• Epitope mapping to identify antibody binding regions:

  • Peptide array analysis to determine specific epitope recognition

  • Comparison with protein sequence databases to identify similar epitopes in other proteins

• Validation experiments:

  • Preabsorption tests with recombinant SPX protein

  • Testing on SPX-knockout or knockdown models (if available)

  • Parallel testing with multiple antibodies targeting different SPX epitopes

• Specificity enhancement strategies:

  • Affinity purification against the immunizing peptide

  • Increasing stringency of washing steps (higher salt concentration)

  • Optimization of blocking reagents (5% BSA often preferred over serum for polyclonal antibodies)

• Analytical confirmation:

  • Western blot analysis with reducing and non-reducing conditions

  • Mass spectrometry confirmation of immunoprecipitated proteins

Cross-reactivity resolution is critical for research validity, especially when studying tissues with complex protein expression patterns .

How can surface plasmon resonance (SPR) be utilized for SPX antibody characterization?

Surface plasmon resonance provides valuable kinetic analysis of SPX antibody-antigen interactions. A methodological approach using high-throughput SPR systems such as "BreviA" enables comprehensive characterization:

• Antibody immobilization procedure:

  • Purify antibody samples via ammonium sulfate precipitation to remove low-molecular-weight components

  • Immobilize diluted solutions on sensors functionalized with nitrilotriacetic acid

  • Establish stable baseline before antigen introduction

• Interaction kinetics measurement:

  • Test with four or five antigen concentrations in fourfold dilution series

  • Implement non-regenerative kinetics methods, avoiding regeneration treatment between measurements

  • Calculate association (kon) and dissociation (koff) rate constants

  • Determine equilibrium dissociation constant (KD) from ratio of koff/kon

• Data analysis and interpretation:

  • Apply appropriate binding models (1:1 Langmuir, heterogeneous ligand, etc.)

  • Compare kinetic parameters across multiple SPX antibody candidates

  • Correlate binding parameters with functional activity in biological assays

This approach enables rapid screening of multiple antibody variants, with complete analysis of 384 interactions possible within a week, facilitating selection of optimal research reagents .

What strategies optimize western blotting protocols for SPX detection?

Optimizing western blot protocols for detecting the 13.3 kDa SPX protein requires methodological rigor:

• Sample preparation considerations:

  • For tissues: homogenization in RIPA buffer with protease inhibitor cocktail

  • For serum/secreted protein: concentration via immunoprecipitation before loading

  • Protein denaturation at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis parameters:

  • 4-12% Bis-Tris gradient gels provide optimal resolution for small proteins

  • MES running buffer (rather than MOPS) improves separation of proteins <20 kDa

  • Include molecular weight markers spanning 5-20 kDa range

  • Load 20-30 μg total protein for tissue lysates

• Transfer optimization:

  • Semi-dry transfer systems at 15V for 30 minutes

  • PVDF membrane with 0.2 μm pore size (rather than 0.45 μm)

  • 10% methanol in transfer buffer to enhance small protein binding

• Detection enhancement:

  • Extended blocking (1-2 hours) with 5% non-fat milk

  • Primary antibody incubation overnight at 4°C at 1:1000-1:2000 dilution

  • High-sensitivity chemiluminescent substrates for detection

  • Longer exposure times may be necessary for low abundance detection

This optimized protocol addresses the challenges associated with detecting small proteins like SPX, which can be lost during standard western blotting procedures .

How should researchers approach quantitative analysis of SPX levels using antibody-based methods?

Quantitative analysis of SPX using antibody-based methods requires careful methodological design:

• ELISA development and validation:

  • Sandwich ELISA design with capture and detection antibodies targeting different epitopes

  • Generation of standard curves using recombinant SPX protein

  • Validation across physiological concentration range (typically pg/mL to ng/mL)

  • Assessment of intra-assay (<10%) and inter-assay (<15%) coefficient of variation

• Sample preparation standardization:

  • Consistent collection protocols (time of day, fasting status for serum)

  • Standardized processing (centrifugation speeds, storage temperatures)

  • Documented freeze-thaw cycles (limit to <3 cycles)

• Normalization strategies:

  • For tissue lysates: total protein determination or housekeeping protein normalization

  • For serum: consideration of diurnal variation patterns

  • For cell culture: cell number or total protein normalization

• Statistical analysis approaches:

  • Appropriate standard curve fitting (4-parameter logistic regression)

  • Lower limit of quantification determination

  • Sample size calculation based on expected effect size

  • Application of appropriate statistical tests based on data distribution

These methodological considerations ensure robust quantitative analysis of SPX across experimental systems and biological samples .

What are common challenges in SPX antibody experiments and their solutions?

Researchers frequently encounter several challenges when working with SPX antibodies. Methodological solutions include:

• Low signal intensity:

  • Increase antibody concentration after titration experiments

  • Extend primary antibody incubation time (overnight at 4°C)

  • Implement signal amplification systems (tyramide signal amplification)

  • Concentrate samples via immunoprecipitation before analysis

  • Verify sample integrity with fresh positive controls

• High background signals:

  • Increase washing stringency (more washes, higher salt concentration)

  • Optimize blocking conditions (test BSA vs. normal serum vs. casein)

  • Pre-absorb polyclonal antibodies with non-specific proteins

  • Reduce secondary antibody concentration

  • Use more specific detection systems

• Inconsistent results between experiments:

  • Standardize sample collection and processing protocols

  • Prepare larger antibody aliquots to avoid freeze-thaw cycles

  • Include internal reference standards in each experiment

  • Document lot numbers and source of all reagents

• Unexpected band patterns in western blots:

  • Verify with multiple antibodies targeting different epitopes

  • Perform peptide competition assays to confirm specificity

  • Consider post-translational modifications or proteolytic processing

  • Run non-reducing gels to assess oligomerization

These systematic troubleshooting approaches address technical variables that may affect experimental outcomes .

How should contradictory findings between SPX antibody applications be reconciled?

When facing contradictory results between different antibody applications (e.g., western blot showing expression but immunohistochemistry negative), methodological reconciliation approaches include:

• Comprehensive technical assessment:

  • Evaluate antibody epitope accessibility in different applications

  • Consider fixation effects on epitope structure (for IHC/IF)

  • Assess protein denaturation effects on epitope recognition

  • Review buffer compatibility across applications

• Biological considerations:

  • Protein localization may affect detection sensitivity

  • Expression levels below detection threshold in some applications

  • Post-translational modifications may alter epitope recognition

  • Splice variants may be recognized differentially

• Validation strategies:

  • Employ orthogonal detection methods (mRNA analysis, mass spectrometry)

  • Test multiple antibodies targeting different epitopes

  • Perform knockdown/knockout validation if feasible

  • Consider reporter systems for low-expression scenarios

• Reconciliation frameworks:

  • Prioritize data from validated applications with proper controls

  • Acknowledge technical limitations in research reports

  • Consider biological context when interpreting conflicting results

  • Design follow-up experiments to specifically address contradictions

What controls are essential when publishing research using SPX antibodies?

Publication-quality research using SPX antibodies requires comprehensive controls:

• Antibody validation controls:

  • Documentation of antibody specificity via western blot

  • Peptide competition/blocking experiments

  • Positive control tissues (pancreas, testis, kidney)

  • Negative control tissues (tissue-specific, based on literature)

  • Isotype control antibodies at matching concentrations

• Technical controls:

  • Secondary antibody-only controls

  • Processing controls (omission of primary antibody)

  • Standardization markers for quantitative comparisons

  • Internal reference standards for inter-assay comparisons

• Biological controls:

  • Physiological manipulation expected to alter SPX expression

  • Time course or dose-response relationships where appropriate

  • Complementary mRNA expression analysis

  • Correlation with functional outcomes

• Reporting requirements:

  • Complete antibody information (source, catalog number, lot, dilution)

  • Detailed methodological description including all control experiments

  • Representative images of controls alongside experimental conditions

  • Quantification methods with statistical analysis

These control standards align with reproducibility initiatives in antibody-based research and enhance publication quality and impact .

How can SPX antibodies be implemented in multiplex detection systems?

Implementation of SPX antibodies in multiplex detection systems requires methodological considerations:

• Antibody compatibility assessment:

  • Cross-reactivity testing between antibody pairs

  • Optimization of antibody concentrations in multiplex format

  • Validation of detection specificity in complex samples

• Multiplex platform selection:

  • Bead-based assays (Luminex) for secreted SPX in biological fluids

  • Multiplex immunofluorescence for tissue localization studies

  • Protein array systems for high-throughput screening

• Protocol optimization:

  • Sequential antibody application to minimize cross-interference

  • Differential fluorophore selection based on spectral separation

  • Optimization of incubation conditions for balanced signal intensity

• Data acquisition and analysis:

  • Establishment of detection thresholds for each target

  • Compensation matrices for spectral overlap

  • Normalization strategies for cross-platform comparisons

  • Multivariate statistical approaches for complex datasets

This methodology enables simultaneous analysis of SPX alongside related hormones or signaling molecules, providing more comprehensive biological insights .

What approaches can identify post-translational modifications of SPX using antibodies?

Identification of post-translational modifications (PTMs) of SPX using antibody-based approaches requires specialized methodologies:

• PTM-specific antibody selection:

  • Phospho-specific antibodies for potential phosphorylation sites

  • Glycosylation-specific detection systems

  • Antibodies recognizing amidated C-terminus

• Enrichment strategies:

  • Immunoprecipitation with pan-SPX antibodies followed by PTM-specific detection

  • Two-dimensional gel electrophoresis to separate modified forms

  • Lectin affinity chromatography for glycosylated forms

• Validation approaches:

  • Treatment with specific enzymes (phosphatases, glycosidases)

  • Mass spectrometry confirmation of enriched fractions

  • Parallel analysis of in vitro modified recombinant SPX

• Functional correlation:

  • Association of PTM status with biological activity

  • Tissue-specific PTM patterns

  • Temporal analysis during physiological responses

Given that SPX is secreted largely as a full-length protein without the signal peptide and not as a hydrolyzed and amidated peptide, these approaches can help elucidate its processing and regulation in different biological contexts .

How should researchers design experiments to study SPX interactions with other proteins?

Studying SPX protein interactions requires careful experimental design:

• Co-immunoprecipitation optimization:

  • Gentle lysis buffer selection to preserve protein complexes

  • Pre-clearing strategies to reduce non-specific binding

  • Antibody orientation considerations (SPX antibody as bait vs. prey)

  • Native elution conditions to maintain complex integrity

• Proximity labeling approaches:

  • BioID or APEX2 fusion protein design

  • Optimization of labeling conditions (biotin concentration, labeling time)

  • Stringent washing protocols to reduce false positives

  • Mass spectrometry analysis of enriched proteins

• Microscopy-based interaction studies:

  • Proximity ligation assay (PLA) optimization

  • FRET/BRET system design for live-cell interaction studies

  • Co-localization analysis with quantitative metrics

  • Super-resolution microscopy for detailed spatial analysis

• Validation strategies:

  • Reciprocal co-immunoprecipitation

  • Domain mapping of interaction interfaces

  • Functional validation of identified interactions

  • Correlation with physiological conditions altering interactions

This methodological framework enables comprehensive characterization of SPX protein interactions, potentially revealing new insights into its biological functions and regulatory mechanisms .