slc18a3b Antibody

What is SLC18A3 and why is it significant in neuroscience research?

SLC18A3 (Solute Carrier Family 18 Member 3), also known as VAChT, is a transmembrane protein that transports acetylcholine into secretory vesicles for release into the extracellular space. This process is critical for proper neuronal function and communication. SLC18A3 plays a crucial role in the cholinergic system, with dysregulation linked to various neurological disorders, including Parkinson's disease and schizophrenia . The protein utilizes a proton gradient established by vacuolar ATPase for acetylcholine transport . Interestingly, the SLC18A3 gene is located within the first intron of the choline acetyltransferase gene , highlighting its close functional relationship with acetylcholine synthesis pathways.

What types of SLC18A3 antibodies are available for research applications?

Several types of SLC18A3 antibodies are available with different specifications:

These antibodies target different epitopes, with many commercial antibodies recognizing amino acids 521-532 or 475-530 in the C-terminal region of the protein . The choice depends on your specific application and experimental design requirements.

What is the molecular weight and structure of SLC18A3 protein?

SLC18A3 is typically detected at approximately 56-57 kDa in Western blot applications. The calculated molecular weight based on its 532 amino acid sequence is around 57 kDa . Product validation data shows the detection of SLC18A3 at:

• ~56 kDa (ABIN1027709)

• 55 kDa (A04891)

The protein is a transmembrane transporter with its C-terminus exposed in the cytoplasmic side of vesicles. The C-terminal region (amino acids 473-532) contains important epitopes targeted by many commercial antibodies . The sequence "GLLTRSRSERDVLLDEPPQGLYDAVRLRERPVSGQDGEPRSPPPGFDACEDDYNYYYRS" represents a common immunogenic region used for antibody production .

How do I select the optimal SLC18A3 antibody for specific research applications?

When selecting an SLC18A3 antibody, consider these critical factors:

Application compatibility:
Different antibodies perform optimally in specific applications:

• For Western blotting: Antibodies validated specifically for WB, such as Boster's Picoband® (A04891), which is optimized for strong signals with minimal background

• For IHC in fixed tissues: Antibodies validated for FFPE tissues or frozen sections

• For multiplexed imaging: Consider conjugated antibodies like Alexa Fluor 647 or APC conjugates

Epitope considerations:

• C-terminal epitopes (aa 475-530 or 521-532) are commonly used and accessible in multiple applications

• Verify epitope conservation if working with non-human species

Species reactivity matrix:

Validation evidence:
Review the validation data provided by manufacturers, particularly Western blot images showing a single band of expected molecular weight and IHC/IF images demonstrating appropriate subcellular localization in known cholinergic regions .

What are the optimal tissue fixation and antigen retrieval methods for SLC18A3 immunodetection?

Fixation and antigen retrieval significantly impact SLC18A3 detection quality:

Recommended fixation protocols:

• For perfusion-fixed tissues: 4% paraformaldehyde (PFA) perfusion followed by 24-48 hour post-fixation at 4°C

• For frozen sections: Brief fixation (10-20 minutes) in 4% PFA after sectioning

• For cultured cells: 4% PFA for 15-20 minutes at room temperature

Antigen retrieval methods:

• Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) for 20 minutes at 95-100°C

• For heavily fixed tissues, consider stronger retrieval methods such as Tris-EDTA (pH 9.0)

• Allow sections to cool slowly to room temperature after HIER for optimal epitope refolding

Permeabilization considerations:

• For IF/ICC: 0.1-0.3% Triton X-100 in PBS for 10-15 minutes

• For brain tissue, longer permeabilization times (20-30 minutes) may be necessary

Always validate your protocol with positive control tissues known to express SLC18A3, such as mouse or rat brain regions with abundant cholinergic neurons (striatum, basal forebrain) .

How can I troubleshoot non-specific binding when using SLC18A3 antibodies?

Non-specific binding is a common challenge when working with SLC18A3 antibodies:

Systematic troubleshooting approach:

• Optimize primary antibody dilution:

  • Test a dilution series (e.g., 1:50, 1:100, 1:200, 1:500)

  • For Western blot: 0.1-0.5 μg/ml typically provides optimal results

  • For IHC/IF: 1:50-1:200 is commonly recommended

• Enhance blocking effectiveness:

  • Increase blocking time to 2 hours or longer

  • Use 5% non-fat milk in TBS for Western blots

  • For IHC/IF, use 5-10% normal serum matching secondary antibody species

  • Add 0.5% BSA to blocking buffer to reduce non-specific protein interactions

• Implement more rigorous washing:

  • Increase wash volume and duration (5-6 washes, 10 minutes each)

  • Add 0.1% Tween-20 to wash buffer for Western blots

  • Use TBS instead of PBS if phospho-epitopes are involved

• Improve negative controls:

  • Include primary antibody omission control

  • Use pre-adsorption with immunizing peptide if available

  • Include tissue/cells known to be negative for SLC18A3

• Address sample-specific issues:

  • For brain tissue with high lipofuscin: Use Sudan Black B treatment

  • For fixed tissues: Optimize antigen retrieval conditions

  • For Western blots: Ensure complete transfer to membrane

What are the best strategies for co-localizing SLC18A3 with other cholinergic markers?

Co-localization studies require careful planning to avoid cross-reactivity and achieve reliable detection:

Optimal marker combinations:

• SLC18A3 + ChAT: Identifies cholinergic neurons and their presynaptic terminals

• SLC18A3 + Synaptophysin: Characterizes cholinergic synaptic vesicle populations

• SLC18A3 + nAChR/mAChR: Examines relationship between transporter and receptors

Technical implementation strategies:

• Antibody selection considerations:

  • Choose primary antibodies from different host species (e.g., rabbit anti-SLC18A3 + mouse anti-ChAT)

  • If using same-species antibodies, consider directly conjugated antibodies or sequential staining

• Fluorophore selection:

  • Use fluorophores with minimal spectral overlap

  • Common combinations: FITC/Alexa488 + Cy3/Alexa555, or Alexa488 + Alexa647

  • Consider tissue autofluorescence in fluorophore selection

• Validation approach:

  • Test each antibody individually before combining

  • Include single-stained controls to establish bleed-through parameters

  • Use Z-stack imaging to confirm true co-localization in three dimensions

Analysis methods:

• For quantitative co-localization, use Pearson's or Mander's coefficients

• For punctate staining patterns, object-based co-localization may be more appropriate

• Consider super-resolution microscopy for fine structural relationships

What is the optimal protocol for SLC18A3 detection by Western blot?

For reliable Western blot detection of SLC18A3, follow this optimized protocol:

Sample preparation:

• Harvest tissue (brain regions rich in cholinergic neurons are ideal) or cells

• Homogenize in RIPA buffer with protease inhibitors

• Sonicate briefly to enhance membrane protein extraction

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

• Collect supernatant and determine protein concentration

SDS-PAGE and transfer:

• Load 20-50 μg total protein per lane on a 10-12% SDS-PAGE gel

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

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

Antibody incubation and detection:

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

• Incubate with primary anti-SLC18A3 antibody (0.5 μg/ml) overnight at 4°C

• Wash 3× with TBS-0.1% Tween, 5 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody (1:10,000) for 1.5 hours

• Wash 3× with TBS-0.1% Tween, 5 minutes each

• Develop using enhanced chemiluminescence detection system

Expected results:
A specific band should be detected at approximately 55-57 kDa, as validated in multiple cell lines including HELA and HEPA cells .

How can I quantify SLC18A3 expression changes in experimental models?

Accurate quantification of SLC18A3 expression requires appropriate normalization and controls:

Western blot quantification:

• Use housekeeping proteins (β-actin, GAPDH) for normalization

• Include concentration standard curves for absolute quantification

• Apply densitometric analysis with software like ImageJ

• Perform statistical analysis across multiple biological replicates

IHC/IF quantification approaches:

• Fluorescence intensity measurement:

  • Capture images under identical acquisition settings

  • Measure mean fluorescence intensity in regions of interest

  • Subtract background from non-specific regions

• Threshold-based analysis:

  • Apply consistent thresholds across all samples

  • Measure percent area above threshold

  • Quantify puncta number and size for synaptic vesicle analysis

• Stereological approaches:

  • Use unbiased stereology for absolute quantification

  • Determine SLC18A3-positive terminal density

  • Apply optical fractionator technique for total number estimation

Experimental design considerations:

• Include appropriate age-matched and treatment controls

• Blind the analyzer to experimental groups

• Validate findings with complementary techniques (e.g., qPCR for mRNA levels)

What controls should be included when validating a new SLC18A3 antibody?

Comprehensive validation requires multiple controls to ensure specificity and reliability:

Essential positive controls:

• Mouse/rat brain lysates for Western blot

• Brain sections containing known cholinergic regions (striatum, basal forebrain)

• Cell lines with confirmed SLC18A3 expression

Critical negative controls:

• Primary antibody omission

• Isotype-matched control antibody

• Non-cholinergic tissues or regions

• SLC18A3 knockout or knockdown samples (if available)

Specificity validation approaches:

• Western blot profile analysis:

  • Confirm single band at expected molecular weight (~56 kDa)

  • Compare against multiple antibodies targeting different epitopes

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Verify signal elimination in Western blot and IHC/IF

• Cross-application validation:

  • Compare staining patterns across multiple techniques

  • Verify subcellular localization is consistent with known biology

Documentation requirements:

• Record all validation experiments with detailed methods

• Document lot number and clone information

• Test multiple biological samples to confirm reproducibility

What are the optimal immunohistochemistry protocols for detecting SLC18A3 in brain tissue?

Brain tissue immunohistochemistry requires specific considerations for optimal SLC18A3 detection:

Protocol for fixed frozen sections:

• Collect fresh brain tissue and fix in 4% PFA (24h at 4°C)

• Cryoprotect in 30% sucrose until tissue sinks

• Freeze in OCT compound and section (20-40 μm thickness)

• Wash sections in PBS (3 × 10 minutes)

• Perform antigen retrieval if needed (citrate buffer pH 6.0)

• Block with 10% normal serum + 0.3% Triton X-100 (2h at RT)

• Incubate with primary anti-SLC18A3 antibody (1:50-1:200) overnight at 4°C

• Wash with PBS (3 × 10 minutes)

• Apply appropriate secondary antibody (1:500, 2h at RT)

• Wash with PBS (3 × 10 minutes)

• Counterstain nuclei (DAPI) and mount with anti-fade medium

Recommendations for FFPE tissue:

• Complete deparaffinization and rehydration

• Extended antigen retrieval (20-30 minutes)

• Longer primary antibody incubation (overnight to 48h at 4°C)

• Consider signal amplification systems for weak signals

Target brain regions:
Focus on cholinergic nuclei for positive signals:

• Nucleus basalis of Meynert

• Striatum

• Pedunculopontine nucleus

• Motor neuron columns in ventral horn of spinal cord

How can researchers analyze antibody binding mechanisms in bivalent binding models?

Understanding the binding mechanisms of antibodies to SLC18A3 can inform experimental design and interpretation:

Mathematical modeling approaches:
Bivalent binding to cell surface receptors can be modeled using ordinary differential equations based on mass action kinetics . This modeling can help predict:

• Antibody concentration effects on target occupancy

• Influence of target density on binding kinetics

• Effects of antibody affinity on detection sensitivity

Experimental validation of binding models:

• Quantitative flow cytometry:

  • Measure receptor occupancy at different antibody concentrations

  • Determine saturation kinetics and apparent Kd values

  • Compare monovalent vs. bivalent binding effects

• Surface plasmon resonance:

  • Measure kon and koff rates

  • Determine affinity constants (KD)

  • Compare different antibody formats (IgG vs. Fab)

Key findings from binding models:

• High-density vs. low-density antigen binding differs significantly

• Bivalent binding can enhance apparent affinity through avidity effects

• When targeting low-abundance proteins like SLC18A3, higher affinity antibodies may not proportionally improve detection if the limiting factor is epitope accessibility

Practical implications:

• For proteins with low expression levels, using signal amplification methods may be more effective than simply increasing antibody affinity

• Consider spatial distribution of target when interpreting binding data

• Account for potential steric hindrances in densely packed synaptic vesicle proteins