SULT1A2 Antibody, HRP conjugated

What is SULT1A2 and what is its role in xenobiotic metabolism?

SULT1A2 (Sulfotransferase 1A2) is a cytosolic enzyme that catalyzes the sulfate conjugation of numerous hormones, neurotransmitters, drugs, and xenobiotic compounds. It belongs to the sulfotransferase family of enzymes that are differentiated by their tissue distributions and substrate specificities. SULT1A2 is one of two phenol sulfotransferases characterized by thermostable enzyme activity. The gene encoding SULT1A2 has two alternatively spliced variants that produce identical proteins with a molecular weight of approximately 34 kDa .

Methodologically, researchers studying SULT1A2's role in metabolism should consider:

• Examining substrate specificity using purified recombinant SULT1A2 and various potential substrates

• Conducting tissue expression profiling to determine localization patterns

• Employing knockout or knockdown models to assess functional consequences of SULT1A2 deficiency

• Comparing activity with other sulfotransferase family members to establish substrate specificity profiles

What are the primary applications for HRP-conjugated SULT1A2 antibodies?

HRP-conjugated SULT1A2 antibodies are primarily utilized in detection-based applications including:

• Western blotting for protein expression analysis

• Immunocytochemistry/immunofluorescence for cellular localization studies

• ELISA for quantitative measurement

• Immunohistochemistry for tissue localization

The direct HRP conjugation eliminates the need for secondary antibodies in detection workflows, reducing background and cross-reactivity issues while streamlining protocols .

What is the significance of HRP conjugation in antibody applications?

HRP conjugation provides several advantages for research applications:

• Direct detection capability without secondary antibodies

• Enhanced sensitivity in enzyme-substrate detection systems

• Excellent stability, with conjugates maintaining 100% activity after storage for up to 2 months at 38°C even at concentrations as low as 0.5 μg/mL

• Compatibility with numerous visualization substrates (DAB, TMB, luminol)

• Simplified experimental workflows with fewer incubation steps

The typical HRP-to-IgG ratio in commercial conjugates is approximately 4:1, which balances detection sensitivity with antibody binding capacity .

How can SULT1A2 antibodies be used to investigate sulfotransferase activity in cancer biology?

Recent research has demonstrated important connections between sulfotransferase activity and cancer progression. While SULT1A1 has been specifically linked to metabolic activation of alkylating agents in liver malignancies including intrahepatic cholangiocarcinoma and hepatocellular carcinoma, similar mechanisms may be relevant for SULT1A2 .

Researchers investigating SULT1A2 in cancer contexts should consider:

• Expression correlation analysis between SULT1A2 levels and cancer progression markers

• Metabolic activation studies examining SULT1A2-dependent drug metabolism

• Development of resistance models through exposure to relevant compounds

• Proteomic analysis to identify changes in SULT1A2 expression across cancer progression

• CRISPR-Cas9 knockout studies to examine functional consequences of SULT1A2 loss in cancer cells

For example, studies with SULT1A1 showed that its expression conferred sensitivity to the compound YC-1, and CRISPR-Cas9-mediated knockout confirmed its functional role in drug response mechanisms . Similar methodologies could be applied to investigate SULT1A2's role in cancer biology.

What considerations are important when validating specificity of SULT1A2 antibodies?

Validating SULT1A2 antibody specificity requires rigorous controls due to the high homology among sulfotransferase family members. Recommended validation approaches include:

• Western blot analysis using recombinant SULT1A2 alongside related family members (SULT1A1, SULT1A3, SULT2A1)

• Testing across multiple species to confirm cross-reactivity claims (human, mouse, rat)

• Testing in SULT1A2 knockout or knockdown models as negative controls

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Peptide competition assays using the immunogen sequence

For SULT1A2 polyclonal antibodies, the immunogen typically corresponds to amino acids 201-295 of human SULT1A2 (NP_001045.1) , or recombinant fusion proteins containing SULT1A2 sequences .

How does sulfotransferase-dependent drug metabolism impact experimental design?

When designing experiments involving SULT1A2:

• Account for potential species differences in sulfotransferase activity

• Consider tissue-specific expression patterns when selecting experimental models

• Evaluate the impact of sulfonation on compound stability and biological activity

• Include appropriate metabolic inhibitors as controls

• Consider the potential for SULT1A2-dependent bioactivation of pro-drugs or xenobiotics

Researchers should particularly note the possibility of SULT1A2-dependent activation of compounds in their experiments, similar to how SULT1A1 has been shown to convert certain compounds to strong alkylators with distinct target profiles .

What are the optimal conditions for Western blotting with HRP-conjugated SULT1A2 antibodies?

Based on the protocol data from multiple sources, the following conditions are recommended for Western blotting with SULT1A2 antibodies:

For HRP-conjugated antibodies specifically, secondary antibodies are not required as the HRP is directly linked to the primary antibody .

What troubleshooting approaches are recommended for non-specific binding?

When encountering non-specific binding with SULT1A2 antibodies, consider these methodological approaches:

• Increase blocking stringency: Extend blocking time or increase blocking agent concentration to 5%.

• Adjust antibody concentration: Use a more dilute antibody solution (1:2000 or higher).

• Modify washing steps: Increase number of washes (5-6 times) and duration (10 minutes each).

• Add detergent: Include 0.1% Tween-20 or 0.1% Triton X-100 in washing and antibody incubation buffers.

• Pre-absorb antibody: Incubate antibody with tissues or cells lacking SULT1A2 to remove cross-reactive antibodies.

• Modify buffer conditions: Adjust salt concentration (150-500 mM NaCl) to reduce non-specific ionic interactions.

• Consider different blocking agents: Test BSA, casein, or commercial blocking agents if milk protein causes problems.

For polyclonal antibodies specifically, affinity purification against the immunogen can reduce non-specific binding, as noted in the antibody specifications .

What are the recommended storage and stability conditions for SULT1A2 HRP-conjugated antibodies?

To maintain optimal activity of SULT1A2 HRP-conjugated antibodies, follow these storage guidelines:

Note that sodium azide, while an effective preservative, can inhibit HRP activity. For working solutions intended for immediate use in HRP-based detection systems, azide-free diluents are recommended .

What quality control metrics should be considered when evaluating SULT1A2 antibody performance?

When evaluating SULT1A2 antibodies for research applications, consider these quality control parameters:

• Purity assessment: Expect >95% purity by SDS-PAGE for affinity-purified antibodies

• Specific activity: Determined by comparing signal intensity to antibody concentration

• Target validation: Confirmation of correct molecular weight (34 kDa for SULT1A2)

• Specificity testing: Cross-reactivity with other sulfotransferase family members should be evaluated

• Lot-to-lot consistency: Compare performance metrics between lots

• Species reactivity: Verify claimed reactivity across human, mouse, and rat samples

• Application suitability: Test in the specific application of interest (WB, ICC/IF, ELISA)

For polyclonal antibodies, batch variation may be greater than for monoclonal antibodies, requiring more extensive validation for each lot .

How should positive and negative controls be designed for SULT1A2 antibody validation?

Robust control design is critical for validating SULT1A2 antibody performance:

Positive Controls:

• Recombinant SULT1A2 protein at known concentrations

• Lysates from cells or tissues with confirmed high SULT1A2 expression (e.g., A431 cells, mouse liver)

• Overexpression systems with tagged SULT1A2 constructs

Negative Controls:

• SULT1A2 knockout cell lines generated via CRISPR-Cas9

• SULT1A2 knockdown samples using validated siRNA/shRNA

• Tissues or cell types with confirmed absence of SULT1A2 expression

• Primary antibody omission controls (for secondary antibody specificity)

• Isotype controls matching the primary antibody's host species and isotype

Specificity Controls:

• Peptide competition using the immunizing peptide or recombinant protein

• Testing against related sulfotransferase family members (SULT1A1, SULT1A3, SULT2A1)

• Comparing multiple antibodies against different epitopes of SULT1A2

What considerations are important when designing multiplexed detection experiments with SULT1A2 HRP-conjugated antibodies?

Multiplexed detection involving SULT1A2 HRP-conjugated antibodies requires careful planning:

• Spectral compatibility: When using multiple detection systems, ensure sufficient separation of emission spectra

• Cross-reactivity prevention: For multi-color immunofluorescence, use antibodies from different host species

• Signal intensity balancing: Adjust antibody concentrations to achieve comparable signal intensities

• Sequential detection: For multiple HRP-conjugated antibodies, consider sequential detection with stripping between rounds

• Chromogenic alternatives: Consider alternative enzymes (AP, β-gal) for truly simultaneous detection

• Tyramide signal amplification: For fluorescent multiplexing, TSA can be used with HRP-conjugated antibodies for sequential detection

When working with HRP-conjugated antibodies specifically, be aware that using multiple HRP-conjugated antibodies simultaneously will result in combined signal that cannot be differentiated unless sequential detection with stripping is employed .

How can SULT1A2 expression be quantified using HRP-conjugated antibodies?

Quantitative analysis of SULT1A2 expression using HRP-conjugated antibodies can be accomplished through several methodological approaches:

• Quantitative Western blotting:

  • Use purified recombinant SULT1A2 to generate a standard curve

  • Include housekeeping protein controls (β-actin, GAPDH) for normalization

  • Apply densitometric analysis using validated software (ImageJ, Image Lab)

  • Ensure signal remains in the linear range of detection

• ELISA-based quantification:

  • Develop a sandwich ELISA using capture and HRP-conjugated detection antibodies

  • Generate standard curves using recombinant SULT1A2 protein

  • Optimize antibody concentrations, incubation times, and blocking conditions

  • Account for matrix effects by preparing standards in the same buffer as samples

• Immunocytochemistry quantification:

  • Use standardized acquisition parameters for microscopy

  • Apply automated intensity measurement across multiple fields

  • Include calibration standards in each experiment

  • Normalize to cell number or area

For all quantitative applications, statistical validation should include assessment of precision (intra- and inter-assay CV%), accuracy (recovery of spiked standards), and linearity within the relevant concentration range .

What strategies can address inconsistent results between different detection methods?

When encountering discrepancies between different detection methods for SULT1A2:

• Epitope accessibility differences:

  • Different sample preparation methods may affect epitope exposure

  • For fixed samples, test alternative fixation methods or antigen retrieval techniques

  • For Western blotting, compare reducing vs. non-reducing conditions

• Method-specific optimization:

  • Each application (WB, IF/ICC, IHC) may require different antibody dilutions

  • Adjust blocking conditions specifically for each methodology

  • Consider application-specific sample preparation requirements

• Cross-validation approaches:

  • Use multiple antibodies targeting different epitopes of SULT1A2

  • Employ orthogonal detection methods (qPCR for mRNA levels, activity assays)

  • Confirm with mass spectrometry-based proteomics approaches

• Quantitative considerations:

  • Establish method-specific limits of detection and quantification

  • Ensure measurements fall within the linear range for each method

  • Account for potential interfering substances specific to each method

When using HRP-conjugated antibodies specifically, remember that conjugation may affect antibody avidity or epitope recognition compared to unconjugated versions of the same antibody .

How do sulfotransferase activity assays complement antibody-based detection approaches?

Activity assays provide functional data that complements the expression information from antibody-based detection:

• Correlation analysis:

  • Compare protein expression levels (by antibody detection) with enzymatic activity

  • Identify post-translational modifications or cofactors affecting activity

  • Distinguish between active and inactive forms of SULT1A2

• Methodological approaches:

  • Radioactive assays using 35S-PAPS (3'-phosphoadenosine-5'-phosphosulfate) as sulfate donor

  • HPLC-based detection of sulfonated products

  • Fluorescent substrate conversion assays

  • Coupled enzyme assays that monitor PAPS consumption

• Activity modulation studies:

  • Test effects of inhibitors on SULT1A2 activity

  • Examine substrate competition effects

  • Assess impact of cellular signaling on enzyme activity

• Species-specific considerations:

  • Compare activity profiles between human, mouse, and rat SULT1A2

  • Account for species differences in substrate specificity

  • Consider tissue-specific activity variations

Activity assays can reveal functional implications of SULT1A2 variants or modifications that may not be apparent from expression analysis alone, such as the role of SULT1A1 in activating compounds like YC-1 in cancer cells .

What are the key considerations when comparing results across different SULT1A2 antibody sources?

When comparing results obtained with different SULT1A2 antibodies:

• Epitope differences:

  • Map the epitope regions targeted by each antibody

  • Different regions may be differentially accessible in various applications

  • Some epitopes may be masked by protein-protein interactions

• Antibody format variations:

  • Compare polyclonal versus monoclonal antibodies

  • Assess differences between various host species (rabbit, mouse, goat)

  • Consider differences in HRP conjugation methods and ratios

• Validation documentation:

  • Review validation data provided by manufacturers

  • Examine knockout/knockdown controls used for validation

  • Check for cross-reactivity testing with related sulfotransferases

• Standardization approaches:

  • Use recombinant protein standards across experiments

  • Maintain consistent sample preparation methods

  • Apply normalization to account for antibody sensitivity differences

• Lot-to-lot variation:

  • For polyclonal antibodies, significant variation may exist between lots

  • Document lot numbers used in critical experiments

  • Consider antibody validation with each new lot

Direct comparison studies should include side-by-side testing of multiple antibodies on identical samples to establish correlation factors and identify potential discrepancies in detection sensitivity or specificity .