MAST3 Antibody, HRP conjugated

What is MAST3 and what is its biochemical function?

MAST3 (Microtubule-associated serine/threonine-protein kinase 3) is a member of the protein kinase superfamily and specifically belongs to the AGC Ser/Thr protein kinase family . This enzyme catalyzes the phosphorylation reaction where ATP + a protein yields ADP + a phosphoprotein . MAST3 is also known by alternate names including KIAA0561 . The protein has a calculated molecular weight of 143 kDa, though it is typically observed at approximately 144 kDa when analyzed via Western blotting . MAST3 functions in signal transduction pathways, as indicated by its classification in research areas .

The full understanding of MAST3's biological role continues to evolve, but its involvement in phosphorylation reactions suggests its importance in cellular signaling cascades and potential regulatory functions in various cellular processes.

What are the key characteristics of commercially available MAST3 Antibody, HRP conjugated?

MAST3 Antibody, HRP conjugated is typically a polyclonal antibody raised in rabbits against recombinant human MAST3 protein fragments . The specific characteristics include:

The HRP conjugation provides direct enzymatic detection capabilities, eliminating the need for secondary antibody incubation steps in various immunoassay protocols .

In which biological samples has MAST3 expression been reliably detected?

MAST3 expression has been validated in multiple biological samples using various detection methods:

This expression profile indicates that MAST3 is present in multiple tissue types across species, with particularly strong expression in neural tissues, suggesting potential specialized functions in the nervous system .

What are the optimal dilution recommendations for different applications of MAST3 antibodies?

When working with MAST3 antibodies, application-specific dilution optimization is critical for reliable results. Based on validated protocols, the following dilution ranges are recommended:

It is strongly recommended to perform antibody titration for each specific experimental system to determine optimal working concentrations. The optimal dilution will depend on factors including target protein abundance, sample type, and detection method sensitivity .

How should researchers design sandwich ELISA experiments for MAST3 detection?

When implementing a sandwich ELISA for MAST3 detection, researchers should follow this methodological approach:

• Capture antibody coating: An antibody specific for MAST3 is pre-coated onto a microplate surface.

• Sample addition: Standards and samples are added to the wells, allowing any MAST3 present to bind to the immobilized antibody.

• Detection antibody addition: After washing away unbound substances, a biotin-conjugated antibody specific for MAST3 is added.

• Enzyme conjugate addition: Following another wash step, Streptavidin-conjugated Horseradish Peroxidase (HRP) is added to the wells.

• Substrate reaction: After a final wash, a substrate solution is added, producing color in proportion to the amount of MAST3 initially bound.

• Signal detection: The color development is stopped, and absorbance is measured using a microplate reader.

The entire working time typically requires 3-5 hours, depending on the operator's experience. For accurate quantitation, a standard curve using purified MAST3 at known concentrations should be prepared alongside experimental samples .

What normalization strategies are available for cell-based MAST3 ELISA assays?

Cell-based ELISA assays for MAST3 require proper normalization to account for well-to-well variations in cell number. Two complementary normalization approaches are recommended:

• GAPDH normalization:

  • Anti-GAPDH antibody is used as an internal positive control.

  • Target (MAST3) absorbance values are normalized to GAPDH signal from the same well.

  • This accounts for variations in cell number and protein expression levels.

• Crystal Violet normalization:

  • Following colorimetric measurement of HRP activity, Crystal Violet whole-cell staining is performed.

  • This staining method provides a measure of total cell density in each well.

  • Absorbance values can then be normalized to cell amounts, adjusting for plating differences.

Using both methods in parallel provides the most robust normalization strategy and enables researchers to detect changes in MAST3 expression independent of cell number variations .

What are the common causes of high background in MAST3 immunodetection assays?

High background in MAST3 immunodetection assays can arise from multiple sources that require systematic troubleshooting:

• Antibody concentration issues:

  • Excessive primary antibody (MAST3 antibody) concentration

  • Recommended solution: Titrate antibody using dilution series (e.g., 1:500, 1:1000, 1:2000, 1:3000)

• Insufficient blocking:

  • Inadequate blocking of non-specific binding sites

  • Recommended solution: Optimize blocking buffer composition and increase blocking time

• Insufficient washing:

  • Residual unbound antibody remaining in the system

  • Recommended solution: Increase wash volume, duration, and number of wash cycles

• Cross-reactivity:

  • Non-specific binding to proteins similar to MAST3

  • Recommended solution: Pre-adsorb antibody with non-target proteins or validate specificity through knockout/knockdown controls

• Sample preparation issues:

  • Excessive protein concentration or non-specific protein interactions

  • Recommended solution: Dilute samples further or modify buffer conditions

When optimizing for Western blot applications specifically, the recommended dilution range of 1:500-1:3000 should be tested to identify the optimal concentration that maximizes specific signal while minimizing background .

How can researchers validate the specificity of MAST3 antibodies for their experimental system?

Validating antibody specificity is critical for reliable MAST3 detection. Multiple complementary approaches should be employed:

• Positive and negative control samples:

  • Positive controls: Use samples with confirmed MAST3 expression (e.g., Jurkat cells, human brain tissue)

  • Negative controls: Use samples known to lack MAST3 expression or tissues from knockout models

• Peptide competition assay:

  • Pre-incubate the MAST3 antibody with purified MAST3 protein or immunogenic peptide

  • Loss of signal in competition samples confirms specificity

• Molecular weight verification:

  • Confirm that the detected band appears at the expected molecular weight (approximately 144 kDa)

  • Multiple or unexpected bands may indicate non-specific binding

• Genetic knockdown validation:

  • Compare detection between wildtype samples and those with MAST3 knocked down via siRNA or CRISPR

  • Reduction in signal proportional to knockdown efficiency confirms specificity

• Cross-species reactivity assessment:

  • Test antibody against samples from multiple species (human, mouse, rat) if cross-reactivity is claimed

  • Confirm consistent detection patterns across species as appropriate

By implementing these validation steps, researchers can establish confidence in the specificity of their MAST3 antibody before proceeding with experimental applications .

How can MAST3 Antibody, HRP conjugated be utilized in multiplex detection systems?

Incorporating MAST3 Antibody, HRP conjugated into multiplex detection systems requires strategic planning:

• Conjugate selection considerations:

  • When multiple proteins are being detected simultaneously, use conjugated antibodies with distinct reporter molecules:

    • MAST3 Antibody, HRP conjugated can be paired with antibodies conjugated to different enzymes (e.g., alkaline phosphatase) or fluorophores

    • This prevents signal overlap in multiplex detection systems

• Substrate selection for HRP detection:

  • TMB (3,3',5,5'-Tetramethylbenzidine): Blue color development, converted to yellow with stop solution

  • DAB (3,3'-Diaminobenzidine): Brown precipitate, compatible with histological counterstains

  • ECL (Enhanced Chemiluminescence): High sensitivity for Western blot applications

  • Select substrate based on compatibility with other detection systems in the multiplex panel

• Sequential detection protocol:

  • First detect MAST3 using the HRP-conjugated antibody

  • Document results completely

  • If necessary, strip membranes or perform inactivation of HRP

  • Proceed with detection of secondary targets using different conjugate systems

• Data normalization strategy:

  • When detecting MAST3 alongside housekeeping proteins (e.g., GAPDH)

  • Normalize MAST3 signal to housekeeping protein signal

  • Account for well-to-well variations in loading or cell number

This approach enables simultaneous or sequential detection of MAST3 alongside other proteins of interest in complex experimental designs .

What are the critical factors for optimizing MAST3 detection in different tissue types?

Detection of MAST3 across various tissue types requires optimization of several parameters:

• Antigen retrieval optimization:

  • For neural tissues (human/mouse/rat brain): Standard retrieval conditions are typically sufficient

  • For human kidney tissue: Two antigen retrieval options have been validated:

    • Primary recommendation: TE buffer pH 9.0

    • Alternative method: Citrate buffer pH 6.0

  • Each tissue type may require empirical optimization of retrieval conditions

• Antibody concentration adjustment by tissue type:

  • Human brain tissue: Start with 1:1000 dilution for Western blot

  • Mouse/rat brain tissue: May require higher antibody concentration (1:500)

  • Human kidney tissue (IHC): Higher antibody concentration recommended (1:20-1:200)

  • Cell lines (Jurkat, HepG2): Standard dilutions typically effective

• Detection method considerations:

  • Western blot: Effective for soluble protein detection across tissue types

  • IHC: Provides spatial context but requires tissue-specific protocol optimization

  • IF/ICC: Effective for subcellular localization in cultured cells

  • Choose method based on research question and tissue type

• Sample preparation variables:

  • Fresh vs. fixed tissue: Protocols should be adjusted accordingly

  • Homogenization methods: Critical for consistent protein extraction

  • Buffer composition: May require tissue-specific optimization

By systematically addressing these variables, researchers can achieve consistent MAST3 detection across diverse tissue types and experimental systems .

How does MAST3 protein expression correlate with signaling pathway activity?

As a serine/threonine kinase involved in signal transduction, monitoring MAST3 expression can provide insights into pathway regulation:

• Experimental design for pathway analysis:

  • Baseline MAST3 expression should be established in resting cells

  • Pathway stimulation experiments can then be conducted with:

    • Growth factors or cytokines that activate relevant signaling cascades

    • Inhibitors targeting upstream or downstream pathway components

    • Time-course analysis to capture dynamic regulation

• Cell-based ELISA approach:

  • The cell-based ELISA format allows detection of MAST3 expression in adherent or suspension cells

  • This method can detect how various stimulation conditions affect MAST3 expression

  • For accurate interpretation, absorbance values should be normalized using either:

    • GAPDH expression as an internal control

    • Crystal Violet staining for cell density normalization

• Data interpretation considerations:

  • Changes in MAST3 expression may reflect:

    • Transcriptional regulation of the MAST3 gene

    • Post-translational modifications affecting antibody recognition

    • Protein stability or turnover alterations

  • These changes should be correlated with functional readouts of pathway activity

• Complementary approaches:

  • Western blot analysis to confirm expression changes

  • Phospho-specific antibodies to assess MAST3 activation status

  • Functional assays to correlate MAST3 expression with kinase activity

By integrating these approaches, researchers can establish meaningful correlations between MAST3 expression patterns and signaling pathway dynamics in their experimental systems .

What are the optimal storage conditions for maintaining MAST3 antibody activity?

Proper storage of MAST3 antibodies is critical for maintaining their specificity and sensitivity over time:

• Temperature conditions:

  • Store MAST3 Antibody, HRP conjugated at -20°C for long-term storage

  • Some manufacturers recommend -80°C for maximum stability

  • Avoid storing at 4°C for extended periods as this can accelerate degradation of the HRP conjugate

• Aliquoting recommendations:

  • Upon receipt, divide the antibody into small working aliquots

  • This minimizes freeze-thaw cycles which can damage both the antibody and the HRP conjugate

  • For small volume products (e.g., 20μl sizes), aliquoting may be unnecessary for -20°C storage

• Buffer considerations:

  • The antibody is typically supplied in a stabilizing buffer containing:

    • 50% Glycerol (serves as a cryoprotectant)

    • 0.01M PBS, pH 7.4 (maintains proper pH)

    • 0.03% Proclin 300 (acts as a preservative)

  • This formulation helps maintain antibody integrity during freeze-thaw cycles

• Stability timeline:

  • When stored properly at -20°C, the antibody is typically stable for one year after shipment

  • Working aliquots can be kept at 4°C for up to one month

  • Avoid repeated freeze-thaw cycles, which significantly reduce antibody performance

• Handling during experiments:

  • Always thaw antibodies on ice

  • Centrifuge briefly before opening to collect solution at the bottom of the tube

  • Return to -20°C storage promptly after use

Following these storage guidelines will help ensure consistent antibody performance across experiments and maximize the useful life of the reagent .

How can researchers assess and monitor the quality of stored MAST3 antibodies over time?

Systematic quality control procedures should be implemented to monitor MAST3 antibody performance:

• Regular performance testing:

  • Run standardized positive control samples (e.g., Jurkat cell lysate)

  • Compare signal intensity and background levels to baseline measurements

  • Document any deviations from expected performance

• Visual inspection protocol:

  • Examine antibody solution for particulates or cloudiness

  • Check for color changes which may indicate contamination

  • Monitor for excessive precipitation which suggests protein denaturation

• Functional testing approach:

  • Compare serial dilutions against historical standard curves

  • Assess detection sensitivity using samples with known MAST3 concentration

  • Evaluate specificity by confirming signal at expected molecular weight (144 kDa)

• Reference standard comparison:

  • Maintain a reference aliquot from each antibody lot

  • Compare performance of working aliquots against reference standard

  • This allows detection of performance degradation over time

• Record-keeping system:

  • Maintain detailed records of antibody performance over time

  • Document lot number, receipt date, aliquoting dates, and experimental outcomes

  • Use this information to establish a replacement schedule based on observed stability

By implementing these quality control measures, researchers can ensure consistent experimental results and determine when antibody reagents should be replaced due to performance degradation .