SMOX Antibody, FITC conjugated

What is SMOX and what biological functions does it serve?

SMOX (Spermine oxidase) is a flavoenzyme that catalyzes the oxidation of spermine to spermidine, playing a crucial role in polyamine homeostasis within cells . It can also utilize N(1)-acetylspermine and spermidine as substrates with varying affinity depending on the isoform and experimental conditions . SMOX contributes significantly to the regulation of intracellular polyamine concentration and potentially influences cellular sensitivity to antitumor polyamine analogs . In Drosophila melanogaster, SMOX is involved in multiple developmental processes including axon guidance, dendrite morphogenesis, determination of adult lifespan, and mushroom body development .

What structural characteristics define the SMOX protein?

SMOX belongs to the dwarfin/SMAD family and contains one MH1 (MAD homology 1) domain and one MH2 (MAD homology 2) domain . The protein has chromatin DNA binding capacity and sequence-specific DNA binding transcription factor activity . In mammals, SMOX is observed at molecular weights between 56-65 kDa, though the calculated molecular weight is approximately 62 kDa . The protein structure enables its enzymatic activity as a polyamine oxidase, contributing to cellular metabolic processes.

What advantages does FITC conjugation provide for SMOX antibody applications?

FITC conjugation provides direct fluorescent visualization of the SMOX protein without requiring secondary antibodies, streamlining immunofluorescence workflows . With excitation at 490nm and emission at 525nm, FITC-conjugated antibodies enable detection in the green spectrum of fluorescence microscopy and flow cytometry . This conjugation strategy is particularly valuable in multi-color immunostaining protocols where distinguishing between different targets is essential. The stable fluorophore attachment maintains signal strength while reducing background issues commonly encountered with indirect detection methods.

What are the recommended applications for SMOX Antibody, FITC-conjugated?

The FITC-conjugated SMOX antibody has been validated for several applications including Enzyme-Linked Immunosorbent Assay (ELISA), Immunoprecipitation (IP), and Western Blot (WB) . While not explicitly stated in all sources, the fluorescent nature of the conjugate also makes it suitable for flow cytometry and immunofluorescence microscopy applications. The antibody demonstrates high specificity against SMOX protein, particularly when used with appropriate controls and optimized protocols for each specific application.

What are the recommended dilutions for different experimental applications?

Based on the technical specifications, the following dilutions are recommended for optimal results:

• ELISA: 1:10,000

• Immunoprecipitation (IP): 1:200

• Western Blot (WB): 1:250

For other applications such as immunofluorescence, researchers should perform titration experiments to determine optimal concentration, typically starting with dilutions in the 1:50-1:500 range . It is essential to optimize antibody concentration for each specific application and sample type to achieve the best signal-to-noise ratio.

What sample preparation techniques are recommended for detecting SMOX with this antibody?

For Western blotting, standard protein extraction protocols using RIPA or NP-40 based lysis buffers are suitable, with care taken to include protease inhibitors to prevent degradation of the target protein . For immunohistochemistry applications, antigen retrieval using TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may serve as an alternative . Proper fixation with 4% paraformaldehyde for cells or formalin for tissues is essential for preserving protein structure while maintaining epitope accessibility. When working with the FITC-conjugated antibody, minimize exposure to light during all steps to prevent photobleaching of the fluorophore.

Which species does the SMOX antibody, FITC-conjugated recognize?

Different SMOX antibody products show varying reactivity profiles. Some FITC-conjugated SMOX antibodies specifically recognize Drosophila melanogaster SMOX protein , while others are designed to detect mouse SMOX protein . For human applications, researchers should select antibodies specifically validated for human SMOX, as indicated in sources that mention human, mouse, and rat reactivity . Cross-reactivity between species should be experimentally verified before proceeding with cross-species studies.

How can researchers verify the specificity of SMOX antibody in their experimental system?

To verify antibody specificity, researchers should implement multiple validation approaches. A critical control is using SMOX knockout/knockdown samples in Western blot or immunostaining experiments to confirm the absence of signal . Comparing the observed molecular weight (56-65 kDa) with the expected size of SMOX protein provides additional validation . Preabsorption tests using the immunizing peptide can further confirm specificity. For FITC-conjugated antibodies, appropriate controls to account for potential autofluorescence and non-specific binding are essential, including isotype controls and secondary-only controls in parallel experiments.

What are the key experimental design considerations when working with polyamine metabolism pathways?

When investigating polyamine metabolism using SMOX antibodies, researchers should consider the dynamic nature of the polyamine pathway and its regulation . Experimental designs should account for potential compensatory mechanisms that may activate when SMOX function is altered. Time-course studies are recommended to capture the dynamic changes in SMOX expression and activity. Additionally, measuring downstream metabolites like spermidine and 3-aminopropanal can provide functional validation of SMOX activity beyond protein expression levels . Considering potential cross-talk with related enzymes like PAOX (polyamine oxidase) is also important for comprehensive pathway analysis.

How can SMOX antibody, FITC-conjugated be used to investigate cancer biology?

SMOX expression has been reported to be elevated in prostate cancer and prostatic intraepithelial neoplasia tissues , making it a potential biomarker for cancer research. For investigating SMOX in cancer biology, researchers can use the FITC-conjugated antibody in multiplex immunofluorescence assays to correlate SMOX expression with other cancer markers. Flow cytometry applications can quantify SMOX expression levels across different cancer cell populations. Live-cell imaging using the FITC-conjugated antibody (in cells engineered for antibody uptake) could potentially track dynamic changes in SMOX localization during cancer progression or in response to treatments targeting polyamine metabolism.

What strategies can overcome common challenges when using FITC-conjugated antibodies in tissue sections?

When working with FITC-conjugated antibodies in tissue sections, researchers frequently encounter autofluorescence issues. To overcome this, treating sections with sodium borohydride (10 mg/ml for 15-30 minutes) or commercial autofluorescence quenching reagents prior to antibody incubation can significantly reduce background fluorescence. Another common challenge is photobleaching during imaging—this can be minimized by using anti-fade mounting media, reducing exposure times, and imaging FITC channels first in multi-channel experiments. For tissues with high melanin content or lipofuscin, additional quenching steps like Sudan Black B treatment (0.1-0.3% in 70% ethanol) may be necessary to improve signal-to-noise ratios when using FITC-conjugated SMOX antibodies.

What approaches can resolve contradictory SMOX expression data between experimental methods?

When researchers encounter discrepancies in SMOX expression data between different techniques (e.g., Western blot vs. immunofluorescence), multiple validation strategies should be employed. First, verify antibody specificity in each application separately using positive and negative controls . Second, consider that post-translational modifications might affect epitope accessibility differently in various techniques. Third, implement alternative detection methods like qRT-PCR to quantify SMOX at the mRNA level. Fourth, use multiple antibodies targeting different epitopes of SMOX to confirm expression patterns. Finally, evaluate experimental conditions that might affect SMOX stability or expression, including cell confluence, serum starvation, or hypoxia, as these factors might explain apparent contradictions in experimental results.

What are the optimal storage conditions for maintaining SMOX antibody, FITC-conjugated activity?

FITC-conjugated SMOX antibodies should be stored at -20°C for long-term storage , with some sources recommending -80°C for extended storage periods . The antibodies are typically supplied in stabilization buffers containing glycerol (often 50%) and may include preservatives like 0.02% sodium azide or 0.03% Proclin 300 . To maintain antibody integrity, avoid repeated freeze-thaw cycles by preparing small working aliquots upon receipt. FITC-conjugated antibodies are particularly sensitive to light exposure, so vials should be wrapped in foil or stored in opaque containers to prevent photobleaching of the fluorophore during storage.

How can researchers assess the quality of SMOX antibody, FITC-conjugated before experimental use?

Before using FITC-conjugated SMOX antibodies in critical experiments, researchers should perform quality control assessments. A simple fluorescence scan (485nm excitation/528nm emission) of the antibody solution can verify that the FITC conjugate remains fluorescently active. Running a dot blot or Western blot with positive control samples (like A549 cells or PC-3 cells known to express SMOX ) can confirm immunoreactivity. For quantitative applications, comparing signal intensity to a standard curve generated with the same antibody lot can ensure consistent performance across experiments. Additionally, checking for aggregation by centrifuging a small aliquot (10,000×g for 5 minutes) can identify potential storage-related quality issues.

What factors should be considered when interpreting fluorescence intensity from FITC-conjugated antibodies?

When analyzing fluorescence intensity data from FITC-conjugated SMOX antibodies, researchers must account for several factors that influence signal output. Photobleaching during imaging can lead to progressive signal decrease, so time-matched acquisition between samples is essential. The pH sensitivity of FITC (optimal fluorescence at pH 8.0-9.0) means that acidic cellular compartments may show reduced fluorescence intensity independent of actual protein concentration. Quantitative comparisons should include standardization controls to normalize for lot-to-lot variations in FITC:antibody ratio. Finally, tissue or cell type-specific autofluorescence in the FITC channel must be carefully subtracted using appropriate background controls to avoid misinterpretation of SMOX expression levels.

How can SMOX antibody be utilized to investigate polyamine metabolism in neurological development?

SMOX plays significant roles in neurological development, particularly in axon guidance, dendrite morphogenesis, and mushroom body development in model organisms like Drosophila . Researchers can use FITC-conjugated SMOX antibodies in neural tissue sections or cultured neurons to track the expression and localization patterns during different developmental stages. Co-staining with neuronal markers can reveal cell type-specific expression patterns. For developmental time-course studies, combining SMOX immunofluorescence with BrdU labeling or other cell cycle markers can correlate SMOX expression with specific phases of neuronal development. The direct fluorescent labeling enables high-resolution confocal imaging to examine subcellular localization in neuronal processes during critical developmental windows.

What methodological approaches can effectively study SMOX in the context of cellular lifespan determination?

Given SMOX's role in determination of adult lifespan , researchers can employ several methodological approaches using FITC-conjugated antibodies. First, comparing SMOX expression levels between young and aged tissue samples via quantitative immunofluorescence can establish age-related expression patterns. Second, knockdown/overexpression studies coupled with live-cell tracking of SMOX using the FITC-conjugated antibody (in permeabilized cells) can directly correlate protein levels with cellular senescence markers. Third, FACS-based approaches can sort cells based on SMOX expression levels (detected via the FITC-conjugated antibody) followed by functional assays measuring cellular lifespan parameters. Finally, high-content screening using automated imaging platforms can assess how pharmacological modulators of longevity pathways impact SMOX expression patterns across large cell populations.

How can researchers integrate SMOX antibody staining with other methodologies to investigate TGF-β signaling pathways?

SMOX is involved in the transforming growth factor beta receptor signaling pathway , making it relevant for studying this important cellular communication system. For integrated studies, researchers can perform dual immunofluorescence using FITC-conjugated SMOX antibodies alongside antibodies against other TGF-β pathway components (labeled with spectrally distinct fluorophores). Phospho-specific antibodies against SMAD proteins can be combined with SMOX detection to correlate activation states. For functional studies, researchers can stimulate cells with TGF-β ligands and track temporal changes in SMOX expression and localization using time-lapse imaging with the FITC-conjugated antibody in compatible cell systems. Proximity ligation assays (PLA) combining the SMOX antibody with antibodies against potential interaction partners can identify novel protein-protein interactions within the TGF-β signaling network.