SMOX Antibody, HRP conjugated

What is SMOX and what is its biological significance?

SMOX (Spermine oxidase) is a flavin-containing amine oxidase that specifically oxidizes spermine. It plays a crucial role in polyamine homeostasis in animal cells and is involved in regulatory mechanisms of cell growth and differentiation. SMOX is also known by several alternative names including PAO, PAOH1, SMO, C20orf16, FLJ20746, and MGC1010 . The gene is identified by HGNC: 15862, OMIM: 615854, and NCBI Gene ID: 54498 .

What applications is SMOX Antibody, HRP conjugated specifically designed for?

The SMOX Antibody, HRP conjugated (product code: CSB-PA021844LB01HU) is specifically optimized for ELISA applications . The horseradish peroxidase (HRP) conjugation allows for direct detection in colorimetric immunoassays without requiring a secondary antibody, making it particularly valuable for quantitative analysis of SMOX in various sample types .

What is the principle behind ELISA assays using SMOX Antibody, HRP conjugated?

SMOX ELISA assays typically employ a sandwich ELISA format where:

• An anti-SMOX antibody is pre-coated onto a microplate

• Samples or standards containing SMOX are added and bind to the immobilized antibody

• A biotin-conjugated detection antibody specific for SMOX is added

• Streptavidin-HRP is added, which binds to the biotin

• TMB substrate is added, which is catalyzed by HRP to produce a blue color that turns yellow after adding stop solution

• The optical density is measured at 450nm, with color intensity proportional to SMOX concentration

What sample types can be analyzed using SMOX antibodies in research settings?

Based on validated ELISA kits for SMOX, appropriate sample types include:

• Serum

• Plasma (both EDTA and heparin)

• Cell culture supernatants

• Other biological fluids

The antibodies have demonstrated reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species .

How can researchers evaluate and optimize the specificity of SMOX Antibody, HRP conjugated?

Evaluating antibody specificity requires multiple approaches:

• Positive and negative controls: Use known SMOX-expressing cell lines (A549, PC-13, MCF-7, HeLa, PC-3) as positive controls

• Cross-reactivity testing: Validate that the antibody does not react with similar proteins or analogues

• Dilution optimization: Test different antibody dilutions to find the optimal signal-to-noise ratio

• Blocking experiments: Use recombinant SMOX protein as a competitive inhibitor to confirm specificity

• Knockdown validation: Compare signal between normal and SMOX-knockdown samples

Commercial SMOX ELISA kits report "no significant cross-reactivity or interference between Human SMOX and analogues" when properly optimized .

What factors affect the reproducibility of experiments using SMOX Antibody, HRP conjugated?

Based on precision data available for SMOX ELISA assays, several factors influence reproducibility:

Intra-assay Precision:

Inter-assay Precision:

To ensure maximum reproducibility, researchers should:

• Use consistent sample preparation methods

• Follow standardized incubation times and temperatures

• Include standard curves in each experiment

• Use the same lot of antibody when possible across experiments

• Implement rigorous quality control measures

How does matrix interference affect SMOX quantification, and what strategies can minimize this interference?

Matrix effects can significantly impact antibody-based detection methods. Recovery data from validated SMOX assays shows:

To address matrix interference:

• Perform sample dilution studies (linearity data shows good recovery at 1:2, 1:4, and 1:8 dilutions)

• Use matrix-matched calibration standards

• Implement sample pre-treatment protocols when necessary

• Consider adding blocking agents specific to the sample type

• Validate recovery in your specific sample matrix by spiking known amounts of SMOX

What considerations should be made when designing multiplex studies that include SMOX Antibody, HRP conjugated?

When incorporating SMOX Antibody, HRP conjugated into multiplex studies:

• Antibody compatibility: Ensure all antibodies used function under similar buffer and pH conditions

• Cross-reactivity assessment: Validate that the SMOX antibody doesn't cross-react with other targets in the multiplex panel

• Signal optimization: Adjust antibody concentrations to achieve comparable signal intensities across all analytes

• Dynamic range alignment: Ensure the detection range for SMOX overlaps with other analytes of interest

• Incubation sequencing: Determine whether sequential or simultaneous incubations provide optimal results

What are the most common causes of high background in ELISA assays using SMOX Antibody, HRP conjugated?

High background in SMOX ELISA assays can stem from multiple sources:

• Insufficient washing: Increase number and duration of wash steps (protocols recommend 2-5 washes depending on the stage)

• Improper blocking: Optimize blocking buffer composition and incubation time

• Antibody concentration: Excessive antibody can cause non-specific binding (dilution optimization recommended)

• Sample contaminants: Ensure samples are properly prepared and free of interfering substances

• Reagent contamination: Use fresh preparation of working solutions (within 30 minutes before assay)

• Temperature effects: Maintain consistent temperature during incubation steps (37°C recommended for SMOX ELISA)

How can researchers address weak or absent signal when using SMOX Antibody, HRP conjugated?

When troubleshooting weak signals:

• Antibody activity: Verify antibody activity has not been compromised during storage (recommended: store at -20°C, stable for one year after shipment)

• Incubation conditions: Ensure proper temperature (37°C) and duration for each step (90 min for sample, 60 min for detection antibody, 30 min for SABC)

• Substrate reaction: Optimize TMB substrate incubation time (10-20 minutes at 37°C)

• Sample concentration: Insufficient target protein may require sample concentration

• Detection system: Verify the HRP detection system is functioning correctly with positive controls

• Antibody dilution: Adjust dilution ratio (biotin-labeled antibody recommended at 1:99, SABC working solution at 1:99)

What factors can affect the stability and shelf-life of SMOX Antibody, HRP conjugated?

Several factors influence antibody stability:

To maximize stability:

• Store concentrated antibody at -20°C when not in use

• Avoid repeated freeze-thaw cycles

• Working solutions should be prepared fresh (within 30 minutes before assay) and cannot be stored for extended periods

• Protect HRP-conjugated antibodies from light exposure

• Use sterile techniques when handling reagents

• Consider adding carrier proteins (like BSA) for dilute antibody solutions

What is the recommended protocol for a sandwich ELISA using SMOX Antibody, HRP conjugated?

The optimized protocol for SMOX detection includes:

• Sample Preparation:

  • Bring all reagents to room temperature before use

  • Prepare standards and samples according to recommended dilutions

• Antibody Preparation:

  • Dilute biotin-labeled detection antibody 1:99 with antibody dilution buffer

  • Dilute SABC (Streptavidin-HRP) 1:99 with SABC dilution buffer

• Assay Procedure:

  • Add 100μl standard or sample to each well

  • Incubate 90 minutes at 37°C

  • Wash plate twice

  • Add 100μl biotin-labeled antibody

  • Incubate 60 minutes at 37°C

  • Wash plate three times (immerse for 1min each)

  • Add 100μl SABC working solution

  • Incubate 30 minutes at 37°C

  • Wash plate five times (immerse for 1min each)

  • Add 90μl TMB substrate

  • Incubate 10-20 minutes at 37°C

  • Add 50μl stop solution

  • Read at 450nm immediately

How should researchers determine the optimal antibody concentration for their specific experimental conditions?

Antibody titration should follow a systematic approach:

• Prepare a series of antibody dilutions (e.g., 1:50, 1:100, 1:200, 1:500, 1:1000)

• Test these dilutions against both positive control samples and negative controls

• Calculate signal-to-noise ratio for each dilution

• Select the dilution that provides the highest specific signal with minimal background

• Validate the chosen dilution with linearity tests using sample dilutions (1:2, 1:4, 1:8)

• Consider the recovery percentages at each dilution:

The recommended starting dilution for SMOX antibodies in IHC applications is 1:50-1:500, while for WB applications 1:2000-1:10000 is suggested . For HRP-conjugated versions in ELISA, follow manufacturer recommendations.

What controls are essential when using SMOX Antibody, HRP conjugated in research applications?

A comprehensive control strategy should include:

• Standard curve controls: Serial dilutions of recombinant SMOX protein

• Positive tissue/cell controls: Confirmed SMOX-expressing samples (A549, PC-13, MCF-7, HeLa, PC-3 cells)

• Negative controls:

  • Primary antibody omission control

  • Isotype control (non-specific rabbit IgG)

  • Known SMOX-negative samples

• Technical controls:

  • Blank wells (no sample, no antibody)

  • Substrate-only wells (to assess spontaneous substrate conversion)

• Sample dilution controls: To confirm linearity of detection

How should researchers interpret quantitative ELISA data for SMOX across different experimental conditions?

Proper interpretation requires consideration of multiple factors:

• Standard curve validation:

  • Confirm R² value >0.98

  • Ensure all sample measurements fall within the linear range

• Normalization considerations:

  • For cell lysates, normalize to total protein concentration

  • For tissue samples, consider using tissue weight or total protein

  • For serum/plasma, adjust for any dilution factors

• Statistical analysis:

  • Account for intra-assay variability (typically 4.8-5.4% CV)

  • Consider inter-assay variability (3.3-5.8% CV) when comparing across experiments

  • Apply appropriate statistical tests based on experimental design

• Biological context:

  • Interpret SMOX levels in context of known polyamine metabolism pathways

  • Consider potential post-translational modifications affecting antibody recognition

How can researchers validate their SMOX detection results across different methodologies?

Cross-validation strategies include:

• Orthogonal methods comparison:

  • Compare ELISA results with Western blot quantification

  • Validate with immunohistochemistry for tissue localization

  • Consider mRNA expression analysis (qPCR) to correlate with protein levels

• Alternative antibody validation:

  • Test multiple antibodies targeting different SMOX epitopes

  • Compare results between different antibody formats (unconjugated vs. HRP-conjugated)

• Functional validation:

  • Correlate SMOX protein levels with enzymatic activity assays

  • Implement knockdown/knockout studies to confirm specificity

• Method-specific controls:

  • For ELISA: recovery and linearity studies

  • For WB: molecular weight confirmation (observed MW: 56-65 kDa)

  • For IHC/IF: peptide competition studies

What approaches can be used to correlate SMOX expression with biological function in complex experimental models?

To establish meaningful correlations:

• Integrative experimental design:

  • Combine quantitative SMOX protein measurement with enzymatic activity assays

  • Monitor changes in polyamine levels alongside SMOX expression

  • Track cellular phenotypes in relation to SMOX expression levels

• Intervention studies:

  • Use SMOX inhibitors to establish causality in observed phenotypes

  • Implement gene silencing (siRNA/shRNA) or overexpression to modulate SMOX levels

  • Consider CRISPR/Cas9 gene editing for complete knockout models

• Contextual analysis:

  • Assess SMOX expression in response to specific stimuli or stressors

  • Monitor SMOX levels during different stages of cell cycle or differentiation

  • Evaluate SMOX expression in pathological versus normal tissues

• Multi-omics integration:

  • Correlate proteomics data including SMOX with transcriptomics

  • Consider metabolomics analysis focusing on polyamine pathway metabolites

  • Apply systems biology approaches to position SMOX within relevant networks