STATH Antibody, HRP conjugated

What is the function of STATH protein and why is it important to study?

Statherin is a phosphoprotein that inhibits calcium phosphate precipitation in saliva, playing a crucial role in maintaining oral calcium homeostasis. It binds to hydroxyapatite surfaces and influences initial bacterial colonization of tooth surfaces . Studying STATH is important for understanding oral biology, calcium regulation mechanisms, and potential biomarker applications in oral diseases. The protein's highly specific C-terminal sequence (YPQPYQPQYQQYTF) serves as a key antigenic determinant for antibody development .

What are the primary applications for STATH Antibody, HRP conjugated?

STATH Antibody, HRP conjugated is primarily used for:

• Western blotting for protein detection and quantification

• Immunohistochemistry for tissue localization studies

• ELISA for quantitative analysis in biological fluids

• Immunocytochemistry for cellular localization studies

The HRP conjugation eliminates the need for secondary antibodies, streamlining experimental workflows similar to other HRP-conjugated antibodies used in research .

What detection methods are compatible with HRP-conjugated antibodies?

HRP-conjugated antibodies can be detected using multiple substrate systems:

ECL systems are particularly effective for Western blot applications, as demonstrated with other HRP-conjugated antibodies in research settings .

What are the optimal storage and handling conditions for maintaining STATH Antibody, HRP conjugated activity?

Based on established protocols for HRP-conjugated antibodies:

• Store at 2-8°C (never freeze, as this degrades HRP activity)

• Maintain in the dark to prevent photobleaching

• Add preservatives (e.g., 50% glycerol with 0.02% sodium azide) for long-term storage

• Avoid repeated freeze-thaw cycles

• Aliquot upon receipt to minimize repeated handling

• Typical shelf life is approximately 6 months when stored properly

How should I determine the optimal working dilution for my specific application?

Determining optimal working dilution requires systematic titration:

• Begin with manufacturer's recommended range (typically 1:1000-1:5000 for Western blotting)

• Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Test all dilutions in parallel with appropriate positive and negative controls

• Evaluate signal-to-noise ratio at each dilution

• Select the dilution providing specific signal with minimal background

• Verify reproducibility with independent experiments

For Western blotting applications, many HRP-conjugated antibodies demonstrate optimal performance at 1:1000-1:4000 dilutions .

What controls should be included when using STATH Antibody, HRP conjugated?

Essential controls include:

Proper controls are essential for meaningful interpretation, particularly when working with complex biological samples like saliva .

How does the HRP conjugation process affect antibody performance compared to unconjugated alternatives?

HRP conjugation introduces several important considerations:

• Direct detection: Eliminates need for secondary antibody, reducing protocol time and potential cross-reactivity

• Signal amplification: Provides enzymatic amplification of detection signal

• Epitope masking: Conjugation can occasionally interfere with antibody binding sites

• Sensitivity: Generally comparable to or slightly less sensitive than two-step detection (primary + secondary-HRP)

• Specificity: Reduces potential cross-reactivity issues from secondary antibodies

• Stability: Less stable than unconjugated antibodies; enzymatic activity may decrease over time

The conjugation typically occurs through covalent attachment to carbohydrate groups or amine residues on the antibody .

What is the mechanism of HRP detection in immunoassays, and how does this influence experimental design?

HRP detection mechanism involves:

• HRP catalyzes the oxidation of substrate (e.g., luminol in ECL systems) by hydrogen peroxide

• Oxidized substrate produces detectable signal (light emission, colored precipitate)

• Signal can be detected by imaging systems (chemiluminescence) or visual inspection (chromogenic)

This mechanism influences experimental design:

• Endogenous peroxidases must be quenched in tissue samples to prevent false positives

• Substrate selection determines sensitivity and signal stability

• Detection method must match substrate (e.g., CCD camera for ECL, brightfield microscopy for DAB)

• Signal development time varies by substrate and must be optimized

How can I optimize sample preparation to preserve STATH epitopes for antibody detection?

Sample preparation optimization:

• Extraction buffer: Use buffers containing phosphatase inhibitors to preserve phosphorylation state of STATH

• Fixation: For tissue/cells, select fixatives (4% paraformaldehyde) that preserve epitope structure

• Antigen retrieval: For IHC applications, test both heat-induced and enzymatic methods to expose masked epitopes

• Denaturation conditions: For Western blotting, optimize reducing/non-reducing conditions based on epitope location

• Storage: Process samples immediately or store at -80°C with protease inhibitors

• Concentration: For dilute samples like saliva, consider concentration methods (TCA precipitation, ultrafiltration)

The C-terminal sequence of STATH (YPQPYQPQYQQYTF) is particularly important for antibody recognition, so preparation methods should preserve this region .

What are common causes of false positives/negatives when using STATH Antibody, HRP conjugated, and how can they be addressed?

Proper experimental controls and validation steps are essential for distinguishing true signals from artifacts .

How should quantitative data from Western blots using STATH Antibody, HRP conjugated be normalized for meaningful comparisons?

For reliable quantitative analysis:

• Include loading controls (housekeeping proteins) on the same membrane

• Use densitometry software to quantify band intensities

• Calculate the ratio of STATH signal to loading control signal for each sample

• Include a standard curve of recombinant STATH protein when absolute quantification is needed

• Run technical replicates (n≥3) to assess variability

• Process all samples for comparison under identical conditions on the same blot

• Apply appropriate statistical tests based on experimental design and data distribution

• Report both normalized values and representative blot images

Proper normalization is crucial for compensating for variations in gel loading, transfer efficiency, and other technical factors .

What strategies can improve detection sensitivity when working with low-abundance STATH in complex samples?

Sensitivity enhancement strategies:

• Sample enrichment: Use immunoprecipitation to concentrate STATH before analysis

• Signal amplification: Implement tyramide signal amplification (TSA) techniques

• Extended exposure: For chemiluminescence, use longer exposure times with low-noise detection

• Substrate selection: Choose high-sensitivity substrates like SuperSignal West Femto for ECL

• Blocking optimization: Test different blocking agents (BSA vs. milk) to improve signal-to-noise ratio

• Antibody concentration: Increase antibody concentration while monitoring background

• Membrane selection: Use PVDF membranes (higher protein binding capacity) instead of nitrocellulose

• Enhanced detection systems: Consider using biotin-streptavidin systems for additional amplification

Combining these approaches can significantly improve detection of low-abundance proteins in complex biological matrices .

How can STATH Antibody, HRP conjugated be integrated into multiplex detection systems?

Multiplex integration strategies:

• Sequential detection: Strip and reprobe membranes for multiple proteins of different molecular weights

• Spectral separation: Combine with antibodies conjugated to different enzymes (e.g., alkaline phosphatase) that use distinct substrates

• Spatial separation: Use on protein arrays or in multi-well formats for parallel detection

• Size-based multiplexing: Detect STATH alongside proteins of different molecular weights on the same blot

• Advanced imaging: Use digital imaging systems that can distinguish subtle differences in signal intensities

When multiplexing, careful optimization is needed to prevent cross-reactivity and ensure balanced signal intensities across targets .

What considerations are important when studying post-translational modifications of STATH using HRP-conjugated antibodies?

Key considerations include:

• Phosphorylation state: STATH contains phosphorylated residues critical to function; phosphatase inhibitors must be included in extraction buffers

• Epitope location: Ensure the antibody epitope doesn't include or isn't masked by the modification of interest

• Modification-specific antibodies: Consider using phospho-specific antibodies alongside total STATH antibody

• Sample preparation: Optimize conditions to preserve labile modifications

• Validation: Confirm modifications with mass spectrometry or other orthogonal methods

• Controls: Include samples treated with modifying/demodifying enzymes (phosphatases, kinases)

The N-terminal region of STATH contains phosphorylated serines that are crucial for its calcium-binding properties .

How can STATH Antibody, HRP conjugated be used in combination with advanced microscopy techniques?

Integration with advanced microscopy:

• STED microscopy: Use HRP-conjugated antibodies with tyramide amplification for super-resolution imaging

• Correlative microscopy: Combine HRP detection (via DAB precipitation) with electron microscopy

• Multi-modal imaging: Use alongside fluorescent probes for complementary structural information

• Live cell applications: Limited due to membrane impermeability; primarily for fixed samples

• Tissue-specific visualization: Combine with histological stains for contextual tissue architecture

• Quantitative imaging: Pair with digital image analysis for quantification of expression patterns

When using for microscopy applications, optimization of signal development time is critical to achieve optimal contrast without excessive background .