SLC15A4 Antibody, HRP conjugated

What are the primary applications for SLC15A4 antibody, HRP conjugated?

SLC15A4 antibodies with HRP conjugation are primarily utilized in Western blot, ELISA, and immunohistochemistry (IHC) applications. The HRP conjugation enables direct detection without secondary antibodies, streamlining experimental workflows. These antibodies are particularly valuable for investigating SLC15A4's role in immune responses and inflammatory processes where it functions as a peptide transporter across membranes . Recommended applications include:

• Western blot: For detecting SLC15A4 protein expression levels in cell lysates

• ELISA: For quantitative measurement of SLC15A4 in biological samples

• Immunohistochemistry: For visualizing SLC15A4 localization in tissue sections

• Immunofluorescence: For cellular localization studies

The typical dilution ranges for these applications are:

• ELISA: 1:2000-1:10000

• IHC: 1:200-1:500

• IF: 1:50-1:200

How should SLC15A4 antibody be validated before experimental use?

Proper validation of SLC15A4 antibodies is essential for ensuring experimental reliability. The recommended validation approach includes:

• Positive controls: Use cell lines known to express SLC15A4 highly, such as THP1 monocytic cells or primary immune cells

• Negative controls: Test in SLC15A4 knockout cells generated through CRISPR-Cas9 technology

• Western blot validation: Confirm a single band at the expected molecular weight (~68 kDa)

• Cross-reactivity assessment: Test against related SLC family members to ensure specificity

• Recombinant protein testing: Use purified SLC15A4 protein as a positive control

Recent studies have successfully validated SLC15A4 antibodies through co-immunoprecipitation (Co-IP) assays with co-expressed SLC15A4 and TASL proteins in HEK293T cells, confirming specific binding interactions .

What is the optimal sample preparation protocol for detecting SLC15A4 in different cell types?

The optimal sample preparation protocol varies by cell type, but generally follows these guidelines:

For immune cells (e.g., macrophages, THP1 cells):

• Lyse 1-5×10⁶ cells in 500μL RIPA buffer supplemented with protease inhibitors

• Include phosphatase inhibitors if studying SLC15A4 phosphorylation status

• Sonicate briefly to shear DNA and reduce sample viscosity

• Centrifuge at 14,000×g for 15 minutes at 4°C to remove debris

• Collect supernatant and determine protein concentration

For tissue samples:

• Homogenize tissue in RIPA buffer (10:1 v/w ratio)

• Incubate on ice for 30 minutes with occasional vortexing

• Centrifuge at 14,000×g for 20 minutes at 4°C

• Collect supernatant for analysis

For antigen retrieval in IHC applications, high-pressure citrate buffer (pH 6.0) has been demonstrated to be effective for SLC15A4 detection in paraffin-embedded tissues .

How can SLC15A4 antibody be used to investigate SLC15A4-TASL interactions in TLR signaling pathways?

Recent structural studies have revealed that SLC15A4 recruits TASL to lysosomes in TLR7/8/9 signaling pathways . To investigate these interactions:

• Co-immunoprecipitation approach:

  • Co-express SLC15A4 and TASL proteins in HEK293T cells

  • Perform pull-down assays using anti-SLC15A4 antibody

  • Analyze precipitated proteins via Western blot with HRP-conjugated antibodies

  • Include wild-type and mutant controls (e.g., SLC15A4 E141K, E465K mutations and TASL Y6A mutation)

• Fluorescence co-localization assay:

  • Express fluorescently-tagged TASL constructs (e.g., TASL(1-20)-EGFP)

  • Co-express with SLC15A4

  • Analyze co-localization via confocal microscopy

  • Quantify correlation coefficients between signals

• Functional validation in immune cells:

  • Generate SLC15A4-null or TASL-null THP1 cells using CRISPR-Cas9

  • Rescue with wild-type or mutant proteins

  • Stimulate with TLR agonists (e.g., R848 for TLR7/8)

  • Measure downstream responses (e.g., IL-8 production)

These approaches can reveal the structural basis of SLC15A4-TASL interaction and how this complex regulates endolysosomal TLR signaling.

What strategies are effective for troubleshooting non-specific binding with SLC15A4 antibodies?

Non-specific binding is a common challenge when working with SLC15A4 antibodies. Effective troubleshooting strategies include:

• Blocking optimization:

  • Try different blocking agents (5% BSA vs. 5% non-fat milk)

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Tween-20 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Test serial dilutions beyond the recommended range

  • For Western blot, start with 1:500 and titrate to 1:5000

  • For IHC, begin with 1:100 and adjust as needed

• Sample preparation refinement:

  • Remove potential interfering proteins via immunodepletion

  • For membrane proteins like SLC15A4, optimize detergent concentrations

  • Consider membrane fraction enrichment techniques

• Validation with knockout controls:

  • Always include SLC15A4-null cells as negative controls

  • Compare staining patterns with published literature

• Cross-reactivity assessment:

  • Pre-absorb antibody with recombinant SLC15A4 protein

  • Test reactivity against related SLC family members

How can SLC15A4 antibodies be utilized to investigate metabolic reprogramming in macrophages?

SLC15A4 has been implicated in mediating M1-prone metabolic shifts in macrophages . To investigate this function:

• Metabolic pathway analysis:

  • Compare wild-type vs. SLC15A4-knockout macrophages using Western blot

  • Analyze key metabolic enzymes like pyruvate dehydrogenase (PDH)

  • Assess downstream metabolites such as acetyl-CoA, itaconate, and transaconitate

• Proximity-dependent biotin identification (BioID) approach:

  • Generate BirA-SLC15A4 fusion constructs

  • Express in macrophages or 293T cells

  • Identify biotinylated proteins via mass spectrometry

  • Analyze enriched pathways (e.g., mTOR signaling, glycolysis)

• Functional metabolic assays:

  • Measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

  • Quantify metabolite levels using mass spectrometry

  • Correlate with SLC15A4 expression levels detected by antibodies

• Signaling pathway integration:

  • Analyze mTORC1 activation status

  • Investigate TFEB nuclear translocation

  • Connect metabolic alterations to inflammatory responses

How can SLC15A4 antibodies be applied to investigate autoimmune disease mechanisms?

SLC15A4 has been implicated in autoimmune diseases, including systemic lupus erythematosus (SLE) and Crohn's disease . Research applications include:

• Tissue expression analysis:

  • Compare SLC15A4 expression in healthy vs. diseased tissues

  • Perform IHC on patient biopsies using HRP-conjugated antibodies

  • Quantify expression differences and correlate with disease severity

• Genetic variant characterization:

  • Identify disease-associated SLC15A4 variants

  • Generate recombinant proteins with these variants

  • Compare antibody reactivity and protein function

• Therapeutic response monitoring:

  • Test SLC15A4 inhibitor effects on protein expression and localization

  • Monitor changes in downstream signaling pathways

  • Correlate with clinical responses in animal models

• Patient sample analysis protocol:

  • Isolate peripheral blood mononuclear cells (PBMCs) from patients

  • Measure SLC15A4 expression via Western blot or flow cytometry

  • Correlate with disease activity indices

  • Test response to TLR agonists ex vivo

Recent studies have shown that small-molecule inhibitors of SLC15A4 can reduce inflammation in mouse models and in cells isolated from lupus patients, highlighting SLC15A4's potential as a therapeutic target .

What methodologies are optimal for studying SLC15A4's role in mast cell biology?

SLC15A4 plays a critical role in mast cell secretory-granule homeostasis . To investigate this function:

• Degranulation assay optimization:

  • Compare wild-type vs. SLC15A4-knockout mast cells

  • Stimulate with IgE/antigen or IL-33

  • Measure degranulation markers (e.g., β-hexosaminidase release)

  • Correlate with inflammatory mediator production

• Secretory granule analysis:

  • Perform electron microscopy to assess granule morphology

  • Use immunofluorescence with SLC15A4 antibodies to determine localization

  • Analyze co-localization with granule markers (e.g., tryptase, chymase)

• Signaling pathway investigation:

  • Analyze mTORC1 activity in wild-type vs. knockout cells

  • Monitor TFEB nuclear translocation

  • Connect these pathways to granule formation and function

• In vivo model applications:

  • Generate mast cell-specific SLC15A4 knockout mice

  • Assess inflammatory responses to various triggers

  • Correlate tissue mast cell phenotypes with systemic responses

How can researchers leverage recent structural insights about SLC15A4 for antibody-based studies?

Recent cryo-electron microscopy studies have revealed three distinct conformational states of SLC15A4 . These insights can inform antibody-based research:

• Conformation-specific antibody applications:

  • Generate antibodies targeting different conformational states

  • Use to track SLC15A4 conformational changes during signaling

  • Investigate how mutations affect protein structure and function

• Interface-targeting strategies:

  • Design antibodies that recognize the SLC15A4-TASL interface

  • Use as tools to block or monitor protein interactions

  • Correlate with downstream signaling events

• Structure-guided epitope selection:

  • Target conserved regions for broad species reactivity

  • Avoid regions involved in conformational changes for stable detection

  • Consider accessibility in native protein conformation

• Therapeutic antibody development:

  • Design antibodies that selectively inhibit disease-associated functions

  • Target specific conformational states or protein-protein interactions

  • Test effects on inflammatory responses in disease models

The structural studies revealed that SLC15A4 undergoes a conformational change from an outward-facing state to an inward-facing state when recruiting TASL, providing new opportunities for targeted antibody development and functional studies .

What controls and validation steps are critical when using SLC15A4 antibodies in proximity ligation assays?

Proximity ligation assays (PLA) can detect protein-protein interactions with high sensitivity. For SLC15A4 studies:

• Essential controls:

  • Positive control: Known SLC15A4 interaction partners (e.g., TASL, RagC)

  • Negative control: Proteins that shouldn't interact with SLC15A4

  • Knockout control: SLC15A4-null cells to verify antibody specificity

  • Mutant controls: SLC15A4 mutations that disrupt specific interactions (E141K, E465K)

• Optimization parameters:

  • Antibody dilutions (typically more dilute than for Western blot)

  • Incubation times and temperatures

  • Washing stringency

  • Signal amplification duration

• Quantification methods:

  • Count PLA spots per cell

  • Measure total fluorescence intensity

  • Analyze subcellular distribution of signals

  • Compare across different experimental conditions

• Validation with complementary techniques:

  • Confirm findings with co-immunoprecipitation

  • Verify with FRET or BiFC approaches

  • Correlate with functional readouts of protein interaction

How can SLC15A4 antibodies be integrated into single-cell analysis workflows?

Single-cell analysis techniques provide unprecedented resolution of cellular heterogeneity. For SLC15A4 research:

• Single-cell Western blot approach:

  • Isolate individual cells from heterogeneous populations

  • Perform microfluidic Western blot with HRP-conjugated antibodies

  • Correlate SLC15A4 expression with cell-specific markers

  • Identify rare cell populations with distinctive expression patterns

• Mass cytometry (CyTOF) integration:

  • Metal-conjugated SLC15A4 antibodies can be incorporated

  • Analyze alongside 30+ other cellular markers

  • Develop high-dimensional profiles of SLC15A4+ cells

  • Correlate with activation states and disease phenotypes

• Single-cell RNA-seq correlation:

  • Sort cells based on SLC15A4 protein levels

  • Perform scRNA-seq on sorted populations

  • Correlate protein expression with transcriptional programs

  • Identify co-regulated pathways and novel interaction partners

• Spatial transcriptomics combination:

  • Use SLC15A4 antibodies for spatial protein mapping

  • Correlate with spatial transcriptomics data

  • Analyze tissue microenvironments and cellular interactions

  • Map SLC15A4 expression to specific tissue niches

What are the considerations for using SLC15A4 antibodies in therapeutic target validation studies?

As SLC15A4 emerges as a potential therapeutic target for autoimmune diseases , antibodies play a crucial role in target validation:

• Expression correlation with disease severity:

  • Quantify SLC15A4 levels in patient samples

  • Correlate with clinical parameters and disease progression

  • Determine if expression changes predict treatment response

  • Evaluate potential as a biomarker

• Target engagement assays:

  • Use antibodies to measure compound binding to SLC15A4

  • Develop cell-based assays for screening inhibitors

  • Quantify protein levels/activity after compound treatment

  • Monitor downstream pathway modulation

• Mechanism of action studies:

  • Determine if therapeutic compounds alter SLC15A4 expression

  • Assess changes in protein localization

  • Evaluate effects on known interaction partners

  • Compare with genetic knockout phenotypes

• Translational research applications:

  • Develop companion diagnostics using validated antibodies

  • Monitor target engagement in clinical samples

  • Assess pharmacodynamic responses in early-phase trials

  • Identify patient subgroups likely to respond to therapy

Recent research has shown that newly developed small molecules targeting SLC15A4 reduced inflammation in lupus models and in cells from lupus patients, demonstrating the potential of SLC15A4 as a therapeutic target .