SUMF2 Antibody

What is SUMF2 and why is it significant in cellular biochemistry?

SUMF2 (Sulfatase Modifying Factor 2) is a critical protein involved in the activation of sulfatases, which are essential enzymes that hydrolyze sulfate esters. SUMF2 plays a crucial role in the post-translational modification process that converts specific cysteine residues to Cα-formylglycine (FGly) in the active site of sulfatases. This modification is essential for sulfatase enzymatic function.

SUMF2 is primarily localized in the endoplasmic reticulum lumen and consists of 301 amino acids. It is expressed widely across human tissues including lung, heart, placenta, brain, liver, pancreas, skeletal muscle, and kidney, indicating its importance in multiple physiological processes . While SUMF2 has low catalytic activity when acting alone, it significantly enhances catalytic efficiency when forming heterodimeric complexes with SUMF1 .

What applications are SUMF2 antibodies commonly used for in research?

SUMF2 antibodies are versatile research tools employed in multiple experimental techniques:

These applications allow researchers to examine SUMF2 expression patterns, protein-protein interactions, and localization in various experimental systems .

How do monoclonal and polyclonal SUMF2 antibodies differ in research applications?

Both monoclonal and polyclonal SUMF2 antibodies are available for research, each with distinct characteristics:

Monoclonal SUMF2 Antibodies:

• Recognize a single epitope on the SUMF2 protein

• Offer high specificity and minimal cross-reactivity

• Provide consistent lot-to-lot reproducibility

• Example: SUMF2 Antibody (D-3), a mouse monoclonal IgG1 kappa antibody that detects human SUMF2

Polyclonal SUMF2 Antibodies:

• Recognize multiple epitopes on the SUMF2 protein

• Provide stronger signal amplification due to multiple epitope binding

• May offer greater detection sensitivity in certain applications

• More tolerant to minor protein denaturation or modifications

• Example: Rabbit polyclonal antibodies against SUMF2 that react with human, mouse, and rat samples

The choice between monoclonal and polyclonal antibodies depends on the specific research requirements, with monoclonals preferred for highly specific detection and polyclonals for applications requiring enhanced sensitivity.

What factors should be considered when selecting an appropriate SUMF2 antibody for specific research applications?

When selecting an SUMF2 antibody, researchers should evaluate several critical factors:

• Target Species Reactivity: Ensure the antibody recognizes SUMF2 from your species of interest. Available SUMF2 antibodies have documented reactivity with human, mouse, and rat SUMF2 .

• Application Compatibility: Verify that the antibody has been validated for your specific application (WB, IP, IHC, IF, ELISA). Some antibodies perform well in multiple applications, while others may be optimized for specific techniques .

• Epitope Location: Consider whether the antibody targets specific domains of SUMF2 that may be relevant to your research question, particularly if studying protein interactions or post-translational modifications.

• Antibody Format: Determine whether you need unconjugated antibodies or those conjugated to specific labels (HRP, PE, FITC, Alexa Fluor® conjugates) based on your detection system .

• Validation Data: Review available validation data including Western blot images, IHC staining patterns, and specificity tests to ensure the antibody performs as expected .

• Lot-to-Lot Consistency: For long-term studies, consider antibodies with documented lot-to-lot consistency to ensure reproducible results.

What are the optimal protocols for using SUMF2 antibodies in Western blotting applications?

For optimal Western blotting with SUMF2 antibodies, follow this methodological approach:

Sample Preparation:

• Extract proteins from cells/tissues using appropriate lysis buffers

• Load 20-30 μg protein per lane (adjust based on SUMF2 expression levels)

• Include appropriate positive controls (e.g., A431 cells show good SUMF2 expression)

Electrophoresis and Transfer:

• Use 5-20% SDS-PAGE gels

• Run at 70V (stacking gel)/90V (resolving gel) for 2-3 hours

• Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

Blocking and Antibody Incubation:

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Incubate with primary SUMF2 antibody at recommended dilution (typically 0.5-2 μg/mL) overnight at 4°C

• Wash with TBS-0.1% Tween (3 times, 5 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody (typically 1:5000 dilution) for 1-1.5 hours at room temperature

Detection:

• Develop using enhanced chemiluminescent detection (ECL) kit

• Expected band size for SUMF2 is approximately 34 kDa

• Multiple bands may be observed due to different glycosylated forms of SUMF2

Troubleshooting Tips:

• If background is high, increase blocking time or try different blocking agents

• If signal is weak, increase antibody concentration or extend incubation times

• For multiple bands, verify specificity with appropriate controls or knockdown experiments

What are the key considerations for using SUMF2 antibodies in immunohistochemistry?

For successful immunohistochemistry (IHC) with SUMF2 antibodies:

Tissue Preparation and Antigen Retrieval:

• Use paraffin-embedded or frozen tissue sections (4 μm thickness recommended)

• Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0) for optimal results

• Alternative: citrate buffer (pH 6.0) may also be effective for some antibodies

Blocking and Antibody Application:

• Block with 10% goat serum to reduce non-specific binding

• Apply SUMF2 primary antibody at appropriate dilution (typically 1:50-1:500 or 2 μg/mL)

• Incubate overnight at 4°C for optimal antigen binding

Detection System:

• Use appropriate secondary antibody system (e.g., peroxidase-conjugated goat anti-rabbit IgG)

• Incubate for 30 minutes at 37°C

• Develop using DAB (3,3'-diaminobenzidine) as chromogen

• Counterstain with hematoxylin for nuclear visualization

Scoring and Interpretation:

• Evaluate staining intensity (0-4 scale: 0=no staining, 4=strongest immunoreactivity)

• Assess percentage of immunoreactive cells (0-4 scale: 0=no positive cells, 4≥75% positive cells)

• Calculate composite score by multiplying intensity and percentage scores

Positive Control Tissues:
SUMF2 antibodies have shown positive IHC staining in:

• Human breast cancer tissue

• Human colon adenocarcinoma tissue

• Human ovarian tumor tissue

• Human placenta tissue

• Human squamous cell carcinoma tissue

How can researchers verify the specificity of SUMF2 antibody staining in their experiments?

Verifying SUMF2 antibody specificity is crucial for experimental validity. Implement these methodological approaches:

• Include Multiple Controls:

  • Positive controls: Use tissues/cells known to express SUMF2 (e.g., A431 cells, human skin tissue)

  • Negative controls: Include samples where the primary antibody is omitted

  • Isotype controls: Use non-specific antibodies of the same isotype to identify non-specific binding

  • Blocking peptide controls: Pre-incubate antibody with the immunizing peptide to confirm epitope-specific binding

• Knockdown/Knockout Validation:

  • Perform siRNA knockdown or CRISPR/Cas9 knockout of SUMF2

  • Compare staining patterns between wild-type and SUMF2-depleted samples

  • Significant reduction in signal in depleted samples confirms specificity

• Multiple Antibody Validation:

  • Use two or more antibodies that recognize different epitopes of SUMF2

  • Concordant results from different antibodies support specificity

• Correlation with mRNA Expression:

  • Compare protein detection patterns with SUMF2 mRNA expression by RT-PCR or in situ hybridization

  • Consistent patterns between protein and mRNA expression support antibody specificity

• Molecular Weight Verification:

  • Confirm the detected band in Western blots appears at the expected molecular weight (34 kDa for SUMF2)

  • Be aware that post-translational modifications may alter the apparent molecular weight

What common issues might researchers encounter when working with SUMF2 antibodies and how can these be resolved?

Researchers working with SUMF2 antibodies may encounter several technical challenges:

For immunohistochemistry specifically, SUMF2 antibody staining has been successfully demonstrated in multiple tissue types with proper antigen retrieval and optimization .

How should researchers interpret differences in SUMF2 antibody staining patterns across different tissue types?

When analyzing SUMF2 staining patterns across tissues, consider these methodological interpretation principles:

• Baseline Expression Patterns:

  • SUMF2 is widely expressed in human tissues including lung, heart, placenta, brain, liver, pancreas, skeletal muscle, and kidney

  • Expect variation in expression levels between tissue types based on physiological functions

• Subcellular Localization Analysis:

  • SUMF2 is primarily localized in the endoplasmic reticulum lumen

  • Evaluate whether staining patterns match the expected subcellular localization

  • Variations in subcellular localization may indicate tissue-specific functions or protein interactions

• Pathological vs. Normal Tissue Comparison:

  • SUMF2 antibodies have shown positive staining in various cancer tissues including breast cancer, colon adenocarcinoma, ovarian tumors, and squamous cell carcinoma

  • Compare expression patterns between normal and pathological tissues of the same origin

  • Quantify differences using standardized scoring systems (intensity and percentage of positive cells)

• Context-Specific Expression:

  • Consider the biological context and disease state when interpreting staining patterns

  • In asthma research, for example, SUMF2 has been shown to interact with IL-13 and inhibit its secretion

  • Evaluate co-expression with interacting partners (e.g., SUMF1, sulfatases) for functional interpretation

• Technical Considerations:

  • Different tissue types may require optimization of antigen retrieval methods

  • Fixation methods and tissue processing can affect epitope availability

  • Standardize your protocol for each tissue type after initial optimization

How can SUMF2 antibodies be utilized to study protein-protein interactions between SUMF2 and its binding partners?

SUMF2 forms important interactions with SUMF1 and sulfatases. These interactions can be studied using SUMF2 antibodies through these methodological approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use SUMF2 antibodies to immunoprecipitate SUMF2 and associated proteins

  • Western blot analysis of immunoprecipitates with antibodies against suspected binding partners

  • This approach has successfully demonstrated SUMF2 interactions with SUMF1 and sulfatases

  • Protocol recommendation: Use 0.5-4.0 μg of SUMF2 antibody for 1.0-3.0 mg of total protein lysate

• Proximity Ligation Assay (PLA):

  • Employ SUMF2 antibodies alongside antibodies against potential binding partners

  • PLA provides in situ visualization of protein interactions with high sensitivity

  • Especially useful for studying endogenous SUMF2 interactions in their native cellular context

• Fluorescence Resonance Energy Transfer (FRET):

  • Use fluorophore-conjugated SUMF2 antibodies (e.g., FITC or Alexa Fluor® conjugates)

  • Particularly valuable for studying dynamic interactions between SUMF2 and binding partners

  • Can provide spatial and temporal information about interaction events

• Pull-down Assays with Recombinant Proteins:

  • Use tagged recombinant SUMF2 proteins for pull-down assays

  • Verify interactions with SUMF2 antibodies in Western blot analysis

  • Has been used to demonstrate that SUMF2 inhibits the enhancing effects of SUMF1 on sulfatases

• Bimolecular Fluorescence Complementation (BiFC):

  • Combine with SUMF2 antibody staining to validate protein-protein interactions

  • Useful for confirming the specificity of observed interactions

Research has shown that SUMF2 forms homodimers and heterodimers with SUMF1, and these interactions can be detected using appropriate antibodies in various experimental systems .

What strategies can be employed to investigate the role of SUMF2 in disease models using SUMF2 antibodies?

SUMF2 has been implicated in various disease processes, particularly in allergic inflammation and sulfatase-related disorders. Here are methodological approaches to investigate SUMF2 in disease models:

• Expression Analysis in Disease Tissues:

  • Use SUMF2 antibodies for IHC or IF to quantify expression in normal versus disease tissues

  • Implement standardized scoring systems to evaluate staining intensity and distribution

  • Compare SUMF2 expression with disease markers and clinical parameters

• Animal Model Studies:

  • In asthma research, SUMF2 expression has been studied in ovalbumin-induced rat models

  • Use SUMF2 antibodies to track protein expression changes during disease progression

  • Correlate SUMF2 levels with disease markers (e.g., IL-13 levels in asthma models)

  • Protocol example: For rat models, use SUMF2 polyclonal antibodies at 1:100 dilution for IHC

• Cell-Specific Expression Analysis:

  • Combine SUMF2 antibody staining with cell-type markers for co-localization studies

  • Determine which cell populations alter SUMF2 expression in disease states

  • Particularly relevant for inflammatory conditions where multiple cell types are involved

• Functional Inhibition Studies:

  • Use SUMF2 antibodies to block protein function in cell culture models

  • Evaluate downstream effects on sulfatase activities and related pathways

  • Assess changes in interacting partners like SUMF1 and IL-13

• Therapeutic Response Monitoring:

  • Track changes in SUMF2 expression or localization following treatment interventions

  • Correlate SUMF2 levels with treatment efficacy and disease outcomes

Research has shown that SUMF2 interacts with IL-13 and inhibits its secretion in bronchial smooth muscle cells, which may be relevant to asthma pathogenesis .

How can researchers utilize SUMF2 antibodies to investigate the regulatory mechanisms controlling SUMF2 and sulfatase activities?

The complex regulation of sulfatase activities involves SUMF2 interactions with SUMF1 and sulfatases. SUMF2 antibodies can help elucidate these mechanisms through:

• Monitoring SUMF1-SUMF2 Complex Formation:

  • Use co-immunoprecipitation with SUMF2 antibodies to isolate SUMF1-SUMF2 complexes

  • Quantify changes in complex formation under different cellular conditions

  • Evidence shows SUMF2 inhibits the enhancing effects of SUMF1 on sulfatases through direct interaction

• Assessing Post-Translational Modifications:

  • Employ SUMF2 antibodies in combination with modification-specific antibodies

  • Track changes in SUMF2 glycosylation states, which appear as multiple bands in Western blots

  • Correlate modifications with functional changes in sulfatase activation

• Subcellular Localization Studies:

  • Use SUMF2 antibodies for immunofluorescence to track protein localization

  • Monitor changes in localization in response to cellular stressors or signaling events

  • SUMF2 is primarily localized in the endoplasmic reticulum lumen, where it participates in sulfatase activation

• Quantitative Analysis in Overexpression/Knockdown Systems:

  • Create experimental systems with altered SUMF2 levels

  • Use SUMF2 antibodies to confirm expression changes

  • Measure corresponding effects on sulfatase activities

  • Research has shown that high levels of SUMF2 decrease the activities of endogenous sulfatases, an effect rescued by SUMF1

• In vitro Reconstitution Assays:

  • Combine purified components (SUMF1, SUMF2, sulfatases)

  • Use SUMF2 antibodies to verify protein presence and interactions

  • Assess functional outcomes on sulfatase activation

A key insight is that SUMF2 modulates sulfatase activities through a direct interaction with both SUMF1 and the sulfatases, forming a regulatory mechanism that can be studied using appropriate antibody-based techniques .

What recent discoveries have been made regarding SUMF2 function using antibody-based techniques?

Recent research utilizing SUMF2 antibodies has revealed several important insights:

• SUMF2 Interactions with Inflammatory Mediators:

  • Studies using yeast two-hybridization and antibody-based confirmation have shown that SUMF2 interacts with IL-13, a key cytokine in allergic inflammation

  • SUMF2 inhibits IL-13 secretion from bronchial smooth muscle cells, independent of IL-13 glycosylation

  • The interaction affects the levels of different forms of intracellular IL-13 (12-kDa vs. 17-kDa)

• SUMF2 Expression in Asthma Models:

  • Investigation of SUMF2 expression in ovalbumin-induced rat models of asthma using antibody-based detection has revealed altered expression patterns

  • Correlation between SUMF2 levels and asthmatic inflammation markers provides insights into potential therapeutic targets

• Regulatory Mechanisms of Sulfatase Activities:

  • Advanced co-immunoprecipitation studies have demonstrated that SUMF2 forms both homodimers and heterodimers with SUMF1

  • SUMF2 can associate with sulfatases with or without SUMF1 present

  • These interactions create a complex regulatory network controlling sulfatase activities

• Structure-Function Relationships:

  • Research using cysteine mutants of SUMF2 (SUMF2(C156A;C290A)) has shown that these residues are critical for SUMF2's inhibitory effect on SUMF1

  • When these cysteines are mutated, SUMF2 no longer inhibits the enhancing effects of SUMF1 on sulfatase activities

• Tissue-Specific Expression Patterns:

  • Immunohistochemical studies using SUMF2 antibodies have revealed expression patterns in various human tissues and cancer samples

  • Detection in breast cancer, colon adenocarcinoma, ovarian tumors, placenta, and squamous cell carcinoma tissues suggests potential roles in cancer biology

How can researchers design experiments to address unresolved questions about SUMF2 function using antibody-based approaches?

Several key questions about SUMF2 remain unresolved. Here are methodological approaches to address these knowledge gaps:

• Question: How does SUMF2 expression change in response to cellular stress?

  • Experimental Design:

    • Subject cells to various stressors (ER stress, oxidative stress, hypoxia)

    • Use SUMF2 antibodies for Western blot and IF to quantify expression changes

    • Correlate with markers of stress response pathways

    • Include time-course analysis to capture dynamic changes

• Question: What is the tissue-specific interactome of SUMF2?

  • Experimental Design:

    • Perform tissue-specific immunoprecipitation with SUMF2 antibodies

    • Combine with mass spectrometry to identify tissue-specific binding partners

    • Validate key interactions with co-immunoprecipitation and proximity ligation assays

    • Compare interactomes between normal and disease states

• Question: How does SUMF2 trafficking between cellular compartments regulate its function?

  • Experimental Design:

    • Use subcellular fractionation followed by Western blotting with SUMF2 antibodies

    • Perform live-cell imaging with fluorescently labeled SUMF2 antibodies

    • Track changes in localization in response to signaling events

    • Correlate with functional outcomes on sulfatase activities

• Question: What is the specific role of SUMF2 in inflammatory diseases beyond asthma?

  • Experimental Design:

    • Analyze SUMF2 expression in tissue samples from various inflammatory conditions

    • Use standardized IHC scoring methods to quantify expression

    • Correlate with disease severity markers and inflammatory cytokines

    • Perform functional studies in relevant cell culture models

• Question: How do post-translational modifications affect SUMF2 function?

  • Experimental Design:

    • Combine immunoprecipitation with SUMF2 antibodies and mass spectrometry

    • Identify specific modification sites and types

    • Generate modification-specific antibodies if available

    • Create mutation constructs to assess functional impacts of modifications

What are the emerging applications of SUMF2 antibodies in therapeutic development and biomarker discovery?

SUMF2 antibodies show promise for several emerging research applications with potential clinical relevance:

• Biomarker Development:

  • SUMF2 expression patterns could serve as diagnostic or prognostic biomarkers in diseases involving sulfatase dysregulation

  • Standardized immunohistochemical scoring of SUMF2 in patient samples may correlate with disease progression or treatment response

  • Multi-marker panels including SUMF2 and interacting partners might provide more comprehensive diagnostic information

• Therapeutic Target Validation:

  • SUMF2 antibodies can help validate this protein as a potential therapeutic target

  • In asthma research, the interaction between SUMF2 and IL-13 suggests potential for intervention in allergic inflammation

  • Blocking or enhancing SUMF2 function could modulate sulfatase activities in conditions where these enzymes are dysregulated

• Drug Mechanism Studies:

  • SUMF2 antibodies can help elucidate the mechanisms of drugs targeting sulfatase pathways

  • Monitor changes in SUMF2 expression, localization, or interactions in response to therapeutic interventions

  • Provide biomarkers for target engagement in clinical trials

• Personalized Medicine Approaches:

  • Evaluate SUMF2 expression patterns in patient samples to guide treatment decisions

  • Identify patient subgroups that might benefit from therapies targeting SUMF2-related pathways

  • Develop companion diagnostics using SUMF2 antibodies for targeted therapies

• Novel Antibody-Based Therapeutics:

  • Development of function-modulating antibodies targeting SUMF2

  • Creation of antibody-drug conjugates for targeted delivery to cells with aberrant SUMF2 expression

  • Engineering of bispecific antibodies targeting SUMF2 and interacting partners simultaneously

The research into SUMF2's interaction with IL-13 in asthma models represents a particularly promising direction, as stated in the literature: "The mechanism of SUMF2 against allergic inflammation requires further study" , indicating an important avenue for future therapeutic development.