SLIT2 Antibody

What is SLIT2 and why is it important in biological research?

SLIT2 (slit homolog 2) is a secreted glycoprotein that functions as a molecular guidance cue in cellular migration, with its effects mediated through interaction with roundabout homolog (ROBO) receptors. Originally identified for its role in axon guidance, SLIT2 has emerged as a multifunctional protein involved in diverse biological processes including immune regulation, tumor suppression, and metabolic control. The protein is encoded by the SLIT2 gene located at chromosome 4p15.2 in humans. SLIT2's importance in research stems from its involvement in multiple physiological and pathological processes, including cancer progression, inflammatory responses, and metabolic regulation .

What are the key characteristics of commercially available SLIT2 antibodies?

SLIT2-specific antibodies, such as the polyclonal antibody 20217-1-AP, are typically generated in rabbits using peptide immunogens. These antibodies demonstrate reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species. The antibodies target specific epitopes of the SLIT2 protein and are available in unconjugated forms. They are typically purified using antigen affinity methods and stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. While the calculated molecular weight of SLIT2 is approximately 170 kDa, researchers often observe bands at 130-140 kDa and 200 kDa in Western blot applications, reflecting potential post-translational modifications or proteolytic processing .

What is the difference between full-length SLIT2 and its proteolytic fragments?

SLIT2 undergoes proteolytic processing to generate biologically active fragments with distinct functions. The full-length SLIT2 protein has a molecular weight of approximately 170-180 kDa, but it is commonly cleaved into several fragments:

• N-terminal SLIT2 (N-SLIT2) fragment: Functions in immune regulation, particularly enhancing neutrophil antimicrobial activity against pathogens like Staphylococcus aureus

• C-terminal SLIT2 fragment (SLIT2-C): A ~50 kDa fragment that promotes adipose thermogenesis and improves glucose homeostasis

This proteolytic processing creates bioactive fragments with specialized functions that may differ from the full-length protein. When using SLIT2 antibodies, researchers should consider whether their antibody recognizes full-length SLIT2, specific fragments, or multiple forms of the protein .

What are the optimal conditions for using SLIT2 antibodies in Western blotting?

For Western blot applications using SLIT2 antibodies, the following methodological approach is recommended:

• Sample preparation: SLIT2 has been successfully detected in HEK-293 cells and mouse brain tissue lysates. Prepare samples using standard lysis buffers containing protease inhibitors.

• Antibody dilution: Use a dilution range of 1:500-1:1000 for optimal results, though this should be titrated for each specific experimental system.

• Expected molecular weights: Prepare to visualize bands at multiple molecular weights:

  • 170 kDa (calculated molecular weight)

  • 130-140 kDa (commonly observed)

  • 200 kDa (possibly reflecting post-translational modifications)

  • 50 kDa (C-terminal fragment)

• Controls: Include positive controls such as HEK-293 cells or mouse brain tissue, and negative controls such as SLIT2 knockout/knockdown samples where available .

Remember that sample-dependent variations may occur, necessitating optimization for your specific experimental system.

How should immunohistochemistry protocols be optimized for SLIT2 detection?

For optimal SLIT2 detection in tissue samples using immunohistochemistry:

• Tissue preparation: SLIT2 has been successfully detected in human kidney tissue and human breast cancer tissue.

• Antigen retrieval: Use TE buffer at pH 9.0 for optimal results. Alternatively, citrate buffer at pH 6.0 may be used, though comparative efficacy should be determined empirically for your tissue of interest.

• Antibody dilution: Use a dilution range of 1:20-1:200, with specific optimization recommended for each tissue type.

• Incubation conditions: Incubate primary antibody overnight at 4°C to maximize specific binding while minimizing background.

• Detection system: Use an appropriate secondary antibody and detection system compatible with rabbit IgG primary antibodies.

• Controls: Include positive control tissues such as human kidney or breast cancer tissue, and negative controls omitting primary antibody .

What methodological considerations are important for immunofluorescence studies with SLIT2 antibodies?

For immunofluorescence applications with SLIT2 antibodies:

• Cell types: HEK-293 cells have been validated for SLIT2 immunofluorescence studies.

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature, followed by permeabilization with 0.1-0.2% Triton X-100.

• Blocking: Block non-specific binding with 5% normal serum (from the species in which the secondary antibody was raised) for 1 hour at room temperature.

• Antibody dilution: Use SLIT2 antibody at 1:50-1:500 dilution, with specific optimization recommended for each cell type and experimental condition.

• Co-staining options: Consider co-staining with subcellular markers to determine the localization pattern of SLIT2, particularly when studying its secretion and processing.

• Controls: Include appropriate negative controls (cells with SLIT2 knockdown) and positive controls (cells overexpressing SLIT2) .

How can SLIT2 antibodies be utilized to investigate SLIT2's role in cancer biology?

SLIT2 has been implicated as a tumor suppressor in lung and breast cancers, with its expression frequently downregulated through promoter hypermethylation and allelic loss. To investigate SLIT2's role in cancer biology using antibodies:

• Expression analysis: Compare SLIT2 protein levels between tumor and normal adjacent tissues using Western blotting and immunohistochemistry. Consider correlation with promoter methylation status and allelic loss at 4p15.2.

• Functional studies: After manipulating SLIT2 expression (overexpression or knockdown), assess phenotypic changes using:

  • Colony formation assays (SLIT2 overexpression suppresses >70% of colony growth in breast tumor lines)

  • Migration and invasion assays (SLIT2 affects cellular migration)

  • Growth inhibition assays using conditioned media containing secreted SLIT2

• Signaling pathway analysis: Investigate SLIT2-ROBO receptor interactions and downstream signaling pathways using co-immunoprecipitation and phosphorylation-specific antibodies.

• In vivo tumor models: Assess tumor growth and metastasis in models with modulated SLIT2 expression, using antibodies for tissue analysis.

This multifaceted approach enables comprehensive investigation of SLIT2's tumor suppressive functions and potential therapeutic applications in cancer .

What approaches can be used to study SLIT2 proteolytic processing and the functions of specific fragments?

To investigate SLIT2 proteolytic processing and the functions of specific fragments:

• Fragment identification:

  • Use Western blotting with antibodies targeting different SLIT2 domains to identify proteolytic fragments in cell culture supernatants or plasma

  • Expected fragments include full-length (~180 kDa), N-terminal fragments, and C-terminal fragments (~50 kDa)

• Fragment isolation:

  • Express tagged versions of SLIT2 (e.g., FLAG-tagged at C-terminus as in Slit2-CTF) for immunoaffinity purification

  • Collect serum-free conditioned media from cells expressing tagged SLIT2 for fragment analysis

• Functional characterization:

  • For the ~50 kDa C-terminal fragment (SLIT2-C): Test effects on adipose thermogenesis, energy expenditure, and glucose homeostasis

  • For N-terminal fragments (N-SLIT2): Assess impact on neutrophil function, including ROS production and granule exocytosis

• In vivo validation:

  • Express specific SLIT2 fragments using viral vectors

  • Measure physiological effects (e.g., metabolic parameters for SLIT2-C, immune responses for N-SLIT2)

This systematic approach enables detailed investigation of the diverse functions of SLIT2 fragments in different biological contexts .

How can researchers investigate SLIT2's role in regulating innate immune responses?

To study SLIT2's role in regulating innate immune responses, particularly in the context of bacterial infections:

• Neutrophil function assays:

  • Reactive oxygen species (ROS) production: Measure using luminol-enhanced chemiluminescence or flow cytometry with ROS-sensitive dyes

  • Degranulation: Assess release of secondary granule contents using ELISA or flow cytometry

  • Bacterial killing: Conduct time-course killing assays with neutrophils exposed to N-SLIT2 and pathogens like S. aureus

• Signaling pathway analysis:

  • Investigate p38 MAPK activation using phospho-specific antibodies

  • Assess NCF1 phosphorylation, a critical component of NADPH oxidase complex

  • Determine the effects of specific pathway inhibitors on SLIT2-enhanced neutrophil functions

• In vivo infection models:

  • Monitor temporal changes in local SLIT2 levels during infection (e.g., skin and soft tissue infection model)

  • Use soluble N-ROBO1 to block endogenous SLIT2 and assess impact on bacterial clearance

  • Evaluate neutrophil recruitment, retention, and activation at infection sites

• Translation to therapeutic applications:

  • Test recombinant SLIT2 fragments as potential anti-microbial therapeutics

  • Investigate synergy with conventional antibiotics

  • Assess efficacy against multiple bacterial pathogens (S. aureus, M. tuberculosis, etc.)

This comprehensive approach enables detailed characterization of SLIT2's immunomodulatory functions and potential therapeutic applications in infectious diseases .

How should researchers address discrepancies in observed molecular weights when detecting SLIT2?

When encountering discrepancies in observed molecular weights for SLIT2:

• Expected pattern: Full-length SLIT2 has a calculated molecular weight of 170 kDa, but researchers commonly observe bands at 130-140 kDa and 200 kDa. Additionally, proteolytic fragments of ~50 kDa may be detected.

• Potential explanations:

  • Post-translational modifications (glycosylation, phosphorylation) can increase apparent molecular weight

  • Proteolytic processing can generate multiple fragments

  • Sample preparation conditions may affect protein integrity

  • Antibody specificity (some antibodies may recognize specific domains or fragments)

• Methodological approach:

  • Use multiple antibodies targeting different SLIT2 domains to confirm band identity

  • Include positive controls (e.g., recombinant SLIT2, SLIT2-overexpressing cells)

  • Employ SLIT2 knockdown/knockout samples as negative controls

  • Consider using reducing vs. non-reducing conditions to assess potential disulfide bonding

• Validation strategies:

  • Immunoprecipitation followed by mass spectrometry to confirm protein identity

  • Expression of tagged SLIT2 constructs with known molecular weights as references

  • Analysis of secreted vs. cellular SLIT2 forms to identify processing patterns

This systematic troubleshooting approach helps ensure accurate interpretation of SLIT2 Western blot data .

What controls should be included when using SLIT2 antibodies in functional studies?

When conducting functional studies with SLIT2 antibodies, include the following controls:

• Expression controls:

  • Positive controls: Cell lines with confirmed SLIT2 expression (e.g., HEK-293 cells, mouse brain tissue)

  • Negative controls: SLIT2 knockdown/knockout samples or cells with naturally low expression

  • Dilution series of recombinant SLIT2 protein for quantitative applications

• Antibody controls:

  • Isotype control antibodies to assess non-specific binding

  • Antibody pre-absorption with immunizing peptide to confirm specificity

  • Secondary antibody-only controls to identify background signal

• Functional validation controls:

  • For tumor suppressor studies: Compare SLIT2-expressing vs. control cells in colony formation assays

  • For neutrophil function: Include both negative control (untreated) and positive control (standard activators like PMA)

  • For in vivo studies: Compare effects of recombinant SLIT2 administration to vehicle control

• Neutralization controls:

  • Use soluble receptors (e.g., N-ROBO1) to block SLIT2 function

  • Compare effects of full-length SLIT2 vs. specific fragments

  • Include dose-response relationships to establish specificity of observed effects

This comprehensive control strategy ensures robust and interpretable results in SLIT2 functional studies .

How can researchers reconcile seemingly contradictory functions of SLIT2 in different biological contexts?

SLIT2 exhibits context-dependent functions that may appear contradictory, particularly regarding cell migration and immune regulation. To reconcile these diverse roles:

• Fragment-specific effects:

  • Different SLIT2 fragments (N-terminal vs. C-terminal) may have distinct or even opposing functions

  • Characterize which fragments predominate in your experimental system using domain-specific antibodies

• Cell type specificity:

  • SLIT2 inhibits neutrophil chemotaxis but enhances bactericidal activity

  • SLIT2 suppresses tumor cell growth but may promote thermogenesis in adipocytes

  • Document cell-specific expression of ROBO receptors and co-receptors that may modify SLIT2 signaling

• Temporal dynamics:

  • In infection models, SLIT2 levels initially decrease (promoting neutrophil recruitment) but later increase (enhancing neutrophil retention and bactericidal activity)

  • Monitor time-course changes in SLIT2 levels and processing during biological processes

• Signaling pathway integration:

  • Map SLIT2 effects on different signaling pathways in various cell types

  • Investigate how SLIT2-ROBO signaling interacts with other pathways (e.g., chemokine receptors, growth factor receptors)

• Methodological approach:

  • Use both loss-of-function (neutralizing antibodies, soluble receptors) and gain-of-function (recombinant proteins) approaches

  • Consider localized vs. systemic effects of SLIT2

This integrated approach helps reconcile seemingly contradictory functions by considering fragment-specific effects, cell type specificity, temporal dynamics, and signaling pathway integration .

What methodological approaches can help investigate SLIT2's potential as an antimicrobial therapeutic?

Recent research suggests SLIT2 may have broad antimicrobial potential. To investigate this promising direction:

• Recombinant protein production:

  • Express and purify specific SLIT2 fragments (particularly N-SLIT2) with confirmed biological activity

  • Verify protein quality using Western blotting, mass spectrometry, and functional assays

• In vitro antimicrobial screening:

  • Test efficacy against diverse pathogens including S. aureus, M. tuberculosis, intestinal pathogens, and viral pathogens

  • Determine minimum inhibitory concentrations and time-kill kinetics

  • Investigate potential synergy with conventional antibiotics

• Mechanism of action studies:

  • Assess direct antimicrobial activity vs. immune-mediated effects

  • For immune-mediated effects, characterize impact on neutrophil ROS production, degranulation, and bacterial killing

  • Map signaling pathways (e.g., p38 MAPK, NCF1 phosphorylation) using phospho-specific antibodies

• In vivo efficacy models:

  • Utilize appropriate infection models (skin and soft tissue infection, pneumonia, etc.)

  • Compare local vs. systemic administration of SLIT2 fragments

  • Monitor bacterial burden, tissue damage, and inflammatory markers

• Pharmacokinetic/pharmacodynamic studies:

  • Determine half-life and tissue distribution of SLIT2 fragments

  • Develop strategies to enhance stability and targeted delivery

  • Assess potential immunogenicity and toxicity

This comprehensive approach will help determine whether SLIT2-based therapeutics could address the growing challenge of antimicrobial resistance .