SLC45A3 Antibody, FITC conjugated

What is SLC45A3 and why is it significant in biomedical research?

SLC45A3 is a proton-associated sucrose transporter that may also transport glucose and fructose. It is a 553-amino acid protein with multiple transmembrane domains . SLC45A3 has gained research significance due to its unique expression profile in normal and cancerous prostatic tissues, making it a valuable biomarker for prostatic carcinoma diagnosis . Recent research has also identified SLC45A3 as a potential therapeutic biomarker for intracerebral hemorrhage-induced white matter injury, where its overexpression may attenuate brain damage . This multifunctionality makes SLC45A3 an important target for both diagnostic and therapeutic research applications.

What is the expression pattern of SLC45A3 in human tissues?

SLC45A3 demonstrates a highly specific expression pattern predominantly in prostatic tissues. Immunohistochemical analyses reveal that SLC45A3 is expressed in both normal prostate and prostatic carcinomas, regardless of grade or metastatic status . Particularly noteworthy is its expression in poorly differentiated prostatic carcinomas, including small cell prostate carcinoma. Studies have demonstrated that SLC45A3 exhibits greater specificity and sensitivity for prostatic carcinomas compared to established markers like PSA (Prostate-Specific Antigen) and PSAP . Single-cell RNA sequencing data indicate that SLC45A3 is also expressed in oligodendrocytes in the brain, suggesting additional roles in the central nervous system .

What experimental applications are FITC-conjugated SLC45A3 antibodies suitable for?

FITC-conjugated SLC45A3 antibodies are primarily optimized for:

• Immunofluorescence microscopy (fixed cell and tissue sections)

• Flow cytometry for cell population analysis

• Fluorescence-activated cell sorting (FACS)

• Confocal microscopy for subcellular localization studies

These antibodies can be used at concentrations of 0.25-2 μg/mL for immunofluorescence applications . The fluorescent properties of FITC (excitation ~495 nm, emission ~519 nm) make these conjugates compatible with standard FITC filter sets on most fluorescence microscopes and flow cytometers.

What protocol is recommended for immunohistochemistry with SLC45A3 antibodies?

For optimal immunohistochemistry results with SLC45A3 antibodies:

• Fix tissue sections in 4% paraformaldehyde

• Perform heat-mediated antigen retrieval using either:

  • Citrate buffer (pH 6.0) or

  • Tris/EDTA buffer (pH 9.0)

• Block with 5% BSA and 0.3% Triton X-100

• Incubate with primary SLC45A3 antibody:

  • For unconjugated antibodies: Use at 1:100-1:2000 dilution depending on the antibody

  • For FITC-conjugated antibodies: Use at 0.25-2 μg/mL

• Incubate overnight at 4°C

• Wash thoroughly with PBS

• For unconjugated antibodies, apply appropriate secondary antibody

• For FITC-conjugated antibodies, proceed directly to counterstaining

• Counterstain as needed (DAPI for nuclei)

• Mount with anti-fade mounting medium to preserve fluorescence

This protocol has been validated on human prostate tissue sections with demonstrable specificity .

How should I optimize Western blotting protocols for SLC45A3 detection?

For Western blotting detection of SLC45A3:

• Prepare protein samples in RIPA lysis buffer

• Load 40 μg protein per lane on 10% SDS-PAGE gels

• Transfer to PVDF membranes

• Block with 5% skim milk at room temperature for 1 hour

• Incubate with primary SLC45A3 antibody at 1:1000 dilution overnight at 4°C

• Wash membranes

• Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature

• Visualize using ECL Plus chemiluminescence

• Quantify bands using ImageJ or similar software

The predicted molecular weight of SLC45A3 is 59 kDa, though observed bands may appear at approximately 55 kDa . When using FITC-conjugated antibodies for other applications, keep in mind that the conjugation may slightly alter the binding characteristics observed in Western blot validation studies.

What controls should be included when working with FITC-conjugated SLC45A3 antibodies?

For rigorous experimental design with FITC-conjugated SLC45A3 antibodies, include:

Positive controls:

• Human prostate tissue or prostate carcinoma samples

• Cell lines with known SLC45A3 expression

Negative controls:

• Isotype control: FITC-conjugated IgG1 (kappa) for mouse monoclonal antibodies or appropriate isotype for other host species

• Secondary antibody only control (for indirect detection methods)

• Tissues known to be negative for SLC45A3 expression

• Blocking peptide competition to confirm specificity

Technical controls:

• Unstained samples to assess autofluorescence

• Single-color controls for spectral overlap compensation in multicolor experiments

• Concentration-matched controls for quantitative comparisons

These controls help distinguish specific SLC45A3 signal from background or non-specific binding.

How can FITC-conjugated SLC45A3 antibodies be used in co-localization studies?

For subcellular co-localization studies with FITC-conjugated SLC45A3 antibodies:

• Select complementary fluorophores with minimal spectral overlap (e.g., TRITC, Cy5)

• Prepare samples using the immunofluorescence protocol outlined above

• For co-staining with MBP (myelin basic protein):

  • After SLC45A3 antibody incubation, wash thoroughly

  • Incubate with MBP antibody (1:500 dilution)

  • Apply appropriate secondary antibody if using unconjugated primary antibodies

• Image using confocal microscopy with sequential scanning to avoid bleed-through

• Analyze co-localization using:

  • Pearson's correlation coefficient

  • Mander's overlap coefficient

  • Line profile analysis across cellular structures

This approach has been successfully used to study SLC45A3 expression in oligodendrocytes and its potential role in white matter injury models .

What is the role of SLC45A3 in gene fusions and how can FITC-conjugated antibodies help study this phenomenon?

SLC45A3 has been identified in recurrent gene fusions in prostate cancer, particularly SLC45A3-ERG fusions. Loss of SLC45A3 protein expression is associated with these gene rearrangements and correlates with unfavorable clinical outcomes . To investigate SLC45A3 fusion events:

• Use FITC-conjugated SLC45A3 antibodies to assess protein expression patterns in tissue samples

• Combine with FISH (Fluorescence In Situ Hybridization) for gene rearrangement detection

• Implement dual immunofluorescence with:

  • FITC-conjugated SLC45A3 antibodies

  • Antibodies against fusion partners (e.g., ERG) labeled with contrasting fluorophores

• Correlate protein expression with clinical parameters

• Consider using multiparametric analysis to integrate protein expression, gene fusion status, and clinical outcomes

This approach provides valuable insights into the relationship between SLC45A3 gene fusions, protein expression, and disease progression in prostate cancer.

How can FITC-conjugated SLC45A3 antibodies be utilized in studying the effects of gene overexpression?

For investigating the effects of SLC45A3 overexpression:

• Establish experimental and control groups:

  • SLC45A3-AAV (adeno-associated virus) overexpression model

  • Scrambled NC-AAV control

• Perform viral transduction using optimized protocols

  • For brain tissue studies: Infuse 3 μl of viral vector (1.21e+12 vg/ml) into the right lateral ventricle

• Allow 3 weeks for expression before experimental interventions

• Use FITC-conjugated SLC45A3 antibodies to:

  • Verify overexpression

  • Track cellular localization

  • Assess effects on tissue morphology

• Quantify protein levels by Western blotting and immunofluorescence intensity

• Correlate with functional outcomes and disease models

This methodology has been successfully applied in intracerebral hemorrhage models, where SLC45A3 overexpression demonstrated neuroprotective effects against white matter injury .

How can I reduce background when using FITC-conjugated SLC45A3 antibodies?

High background is a common challenge with immunofluorescence. To mitigate this issue:

• Optimize antibody concentration through titration experiments

• Increase blocking stringency:

  • Use 5% BSA with 0.3% Triton X-100

  • Consider adding 5-10% serum from the secondary antibody host species

• Extend blocking time to 2 hours at room temperature

• Increase wash duration and frequency (4-5 washes of 5-10 minutes each)

• Prepare antibody dilutions in blocking buffer

• Filter antibody solutions through a 0.22 μm filter to remove aggregates

• If using paraffin sections, ensure complete deparaffinization

• Consider using Sudan Black B (0.1-0.3%) to quench tissue autofluorescence

• Use TBS instead of PBS if phosphate interference is suspected

These optimizations should significantly improve signal-to-noise ratio in your experiments.

Why might SLC45A3 detection be inconsistent between different tissue preparation methods?

Inconsistent SLC45A3 detection may result from various factors:

• Fixation-dependent epitope sensitivity:

  • SLC45A3 epitopes may be differentially preserved in formalin vs. alcohol fixation

  • Optimize fixation duration (typically 24 hours for tissues)

• Antigen retrieval requirements:

  • Heat-mediated antigen retrieval is critical for SLC45A3 detection

  • Different antibody clones may require specific buffer systems:

    • Citrate buffer (pH 6.0)

    • Tris/EDTA buffer (pH 9.0)

• Antibody clone specificity:

  • Clone [EPR4795(2)] recognizes different epitopes than clone [SLC45A3/7648]

  • Each clone may require specific optimization

• Tissue processing variables:

  • Fresh frozen vs. FFPE (formalin-fixed paraffin-embedded) protocols

  • Section thickness (optimal: 8 μm for immunofluorescence)

  • Storage conditions of prepared slides

For consistent results, standardize your tissue preparation protocol and validate antibody performance on known positive controls using the same methodology.

How can FITC-conjugated SLC45A3 antibodies be utilized in prostate cancer research?

FITC-conjugated SLC45A3 antibodies offer several applications in prostate cancer research:

• Diagnostic applications:

  • Differentiation of prostatic from non-prostatic tumors

  • Identification of prostatic origin in metastatic tissues

  • Detection in poorly differentiated carcinomas where other markers may fail

• Prognostic marker investigations:

  • Correlation of expression levels with clinical outcomes

  • Association with SLC45A3-ERG gene rearrangements

  • Comparison with other prostate biomarkers (PSA, PSAP)

• Therapeutic target evaluation:

  • Monitoring SLC45A3 expression in response to treatments

  • Identification of SLC45A3-expressing cell populations

  • Assessment of antibody-drug conjugate targeting potential

Studies have demonstrated that SLC45A3 expression is maintained in most prostate cancers regardless of grade or metastatic status, making it a valuable research tool for tracking prostatic cells throughout disease progression .

What is the significance of SLC45A3 in neurological research and how can FITC-conjugated antibodies contribute?

Recent discoveries highlight SLC45A3's potential in neurological research:

• Role in white matter injury models:

  • SLC45A3 is expressed in oligodendrocytes

  • Overexpression ameliorates brain injury after intracerebral hemorrhage

  • Involves regulation of oligodendrocyte differentiation and fatty acid metabolism

• Research applications of FITC-conjugated SLC45A3 antibodies:

  • Tracking changes in SLC45A3 expression following brain injury

  • Co-localization with oligodendrocyte markers like MBP

  • Monitoring effects of experimental therapies on SLC45A3-expressing cells

• Experimental design considerations:

  • Use stereotactic injection models for localized studies

  • Consider both acute and chronic timepoints (24h to 3 weeks post-injury)

  • Pair with diffusion tensor imaging for comprehensive white matter assessment

These applications represent emerging directions in SLC45A3 research beyond its established role in prostate biology.