SLC51A Antibody, FITC conjugated

What is SLC51A protein and what biological functions does it perform?

SLC51A, also known as Organic solute transporter subunit alpha (OST-alpha), is an essential component of the Ost-alpha/Ost-beta complex. This heterodimeric complex functions as the intestinal basolateral transporter responsible for bile acid export from enterocytes into portal blood . SLC51A partners with SLC51B (OST-beta) to form a functional complex that efficiently transports major species of bile acids, particularly taurocholate . The complex plays an important role in the enterohepatic circulation of bile acids and can also transport steroids such as estrone 3-sulfate and dehydroepiandrosterone 3-sulfate . Additionally, the transporter can mediate the movement of eicosanoids such as prostaglandin E2 . The OST-alpha/OST-beta complex exhibits preferential transport of taurine conjugates compared to glycine-conjugated bile acids across the basolateral membrane .

What are the key specifications of the SLC51A Antibody, FITC conjugated?

The SLC51A Antibody, FITC conjugated is a polyclonal antibody raised in rabbits against a recombinant Human Organic solute transporter subunit alpha protein (amino acids 1-48) . It specifically reacts with human samples and has been validated for ELISA applications . The antibody is of IgG isotype and is conjugated to Fluorescein Isothiocyanate (FITC), making it suitable for fluorescence-based detection methods . It is purified using Protein G with >95% purity . The antibody is provided in liquid form in a buffer containing 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as a preservative . Proper storage requires keeping the antibody at -20°C or -80°C while avoiding repeated freeze-thaw cycles .

What experimental applications are validated for SLC51A Antibody, FITC conjugated?

The SLC51A Antibody, FITC conjugated has been primarily validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications according to multiple sources . While the FITC conjugation suggests its potential utility in fluorescence-based applications such as flow cytometry, immunofluorescence microscopy, and fluorescence-activated cell sorting, these applications may require additional validation by researchers for their specific experimental conditions . Related SLC51A antibodies with different conjugates (such as HRP or Biotin) are also available for specific applications, including ELISA . For investigators interested in exploring immunohistochemistry (IHC) or Western blotting (WB) applications, it would be advisable to conduct preliminary validation studies or consider alternative antibody formats that have been specifically validated for these techniques .

How does the heterodimeric interaction between SLC51A and SLC51B affect antibody selection and experimental design?

The functional unit of SLC51A (OST-alpha) requires interaction with SLC51B (OST-beta) to form the active heterodimeric transporter complex . This interaction introduces important considerations for experimental design. SLC51B has been shown to modulate SLC51A glycosylation, membrane trafficking, and stability activities , suggesting that the detection of SLC51A alone may not fully represent the functional status of the transporter complex.

When designing experiments, researchers should consider whether their research questions focus on SLC51A protein expression, localization, or functional activity of the complete OST-alpha/OST-beta complex. For comprehensive studies, parallel detection of both SLC51A and SLC51B may be necessary. The epitope recognized by this antibody (amino acids 1-48 of SLC51A) should be evaluated for potential masking or conformational changes when SLC51A interacts with SLC51B. Functional assays examining transporter activity should account for the presence and activity of both subunits. Co-immunoprecipitation experiments may be valuable for studying the intact complex, though the FITC conjugation may introduce limitations for this application.

What methodological approaches can be employed to study the role of SLC51A in bile acid transport using this antibody?

To investigate SLC51A's role in bile acid transport using the FITC-conjugated antibody, several methodological approaches can be implemented:

• Cellular Localization Studies: The FITC conjugation makes this antibody suitable for immunofluorescence microscopy to visualize the subcellular localization of SLC51A in intestinal epithelial cell models or tissue sections. This can be particularly valuable for examining whether certain conditions affect membrane trafficking of the transporter.

• Co-localization Analysis: Dual immunofluorescence studies combining this FITC-conjugated SLC51A antibody with antibodies against SLC51B (perhaps conjugated to a different fluorophore) can determine the degree of co-localization of both transporter subunits.

• Expression Correlation with Transport Activity: Flow cytometry using this antibody can quantify SLC51A expression levels in heterogeneous cell populations, which can then be correlated with bile acid transport measurements in the same cells.

• In vitro Transport Assays: Following confirmation of SLC51A expression using this antibody, radiolabeled or fluorescently-labeled bile acid transport assays can be performed to assess functional correlations.

• Competitive Inhibition Studies: The antibody can be used to confirm expression before conducting experiments where competitive inhibitors of bile acid transport are tested against different bile acid species, including taurocholate, which is efficiently transported by the OST-alpha/OST-beta complex .

How can researchers differentiate between monomeric SLC51A and the functional SLC51A/SLC51B heterodimer in experimental systems?

Differentiating between monomeric SLC51A and the functional heterodimer presents a significant challenge in SLC51A research. The following methodological approaches can address this question:

• Size-based Separation: Native gel electrophoresis followed by Western blotting (using non-FITC conjugated anti-SLC51A antibodies) can separate the monomeric from heterodimeric forms based on molecular weight differences.

• Co-immunoprecipitation: Using antibodies against SLC51B to pull down the complex, followed by detection of SLC51A, can confirm the presence of heterodimers.

• Proximity Ligation Assay (PLA): This technique can detect protein-protein interactions in situ by combining antibodies against both SLC51A and SLC51B, followed by a ligation step that only produces a signal when the two proteins are in close proximity.

• Förster Resonance Energy Transfer (FRET): Using the current FITC-conjugated SLC51A antibody as a donor and a compatible fluorophore-conjugated SLC51B antibody as an acceptor, FRET can detect close association between the two proteins.

• Functional Transport Assays: Since the heterodimer is required for transport function, correlation between detected SLC51A expression (using this antibody) and transport activity can indirectly indicate the presence of functional heterodimers.

• Sucrose Gradient Ultracentrifugation: This can separate protein complexes based on their sedimentation coefficients, allowing distinction between monomeric SLC51A and the SLC51A/SLC51B complex.

What are the optimal fixation and permeabilization conditions for immunofluorescence using FITC-conjugated SLC51A antibody?

When performing immunofluorescence using the FITC-conjugated SLC51A antibody, optimal fixation and permeabilization conditions are critical for preserving both epitope accessibility and fluorophore activity. While specific validation data for this particular antibody is limited in the provided information, the following methodological guidelines are recommended based on general practices for FITC-conjugated antibodies detecting membrane proteins:

• Fixation Options:

  • 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature preserves morphology while maintaining most epitopes

  • For the SLC51A epitope (amino acids 1-48) , mild fixation is typically preferable to prevent epitope masking

  • Avoid methanol fixation which can quench FITC fluorescence

• Permeabilization Considerations:

  • Since SLC51A is a membrane protein, gentle permeabilization is recommended

  • 0.1-0.2% Triton X-100 for 5-10 minutes is generally suitable

  • Alternatively, 0.1% saponin may provide gentler permeabilization for maintaining membrane protein structure

• Blocking Conditions:

  • 5-10% normal serum (from species unrelated to the primary antibody) in PBS with 0.1% Tween-20

  • Include 1% BSA to reduce non-specific binding

• Antibody Dilution:

  • Initial testing at 1:50-1:200 dilution range is recommended

  • Titration experiments should be performed to determine optimal signal-to-noise ratio

• Controls:

  • Include a negative control (secondary antibody alone or isotype control)

  • If possible, include a positive control tissue/cell known to express SLC51A

What considerations should be made when designing flow cytometry experiments with this antibody?

Flow cytometry experiments using the FITC-conjugated SLC51A antibody require careful planning and controls:

• Sample Preparation Protocol:

  • For cell suspensions: gentle dissociation methods should be used to preserve membrane proteins

  • Fixation with 1-2% PFA is typically sufficient; stronger fixation may reduce epitope accessibility

  • For intracellular detection, permeabilization with 0.1% saponin is recommended

• Fluorophore Considerations:

  • FITC excites at 494 nm and emits at 520 nm (green spectrum)

  • Consider spectral overlap if using multiple fluorophores

  • FITC is susceptible to photobleaching; minimize light exposure during sample preparation

• Critical Controls:

  • Unstained cells to establish autofluorescence baseline

  • Isotype control (FITC-conjugated rabbit IgG) to determine non-specific binding

  • Positive control (cell line with confirmed SLC51A expression)

  • Single-color controls if performing multicolor flow cytometry

• Titration Experiments:

  • Perform antibody titration to determine optimal concentration

  • Test range: 0.1-10 μg/ml based on typical antibody usage

• Analysis Considerations:

  • Gating strategy should account for cell size/viability

  • Analyze the percentage of positive cells and mean fluorescence intensity

  • For membrane proteins, surface expression may have biological significance distinct from total protein levels

How can researchers validate the specificity of this antibody for their particular application?

Validating antibody specificity is crucial for generating reliable scientific data. For the SLC51A FITC-conjugated antibody, researchers should consider the following validation approaches:

• Positive and Negative Control Samples:

  • Use cells/tissues with known high expression of SLC51A (intestinal epithelial cells, hepatocytes) as positive controls

  • Use cells with confirmed absence of SLC51A or SLC51A-knockout models as negative controls

• Gene Knockdown/Knockout Validation:

  • Compare staining between wild-type samples and those with SLC51A knocked down using siRNA or CRISPR-Cas9

  • Expected result: Significant reduction in signal in knockdown/knockout samples

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide (amino acids 1-48 of SLC51A)

  • Expected result: Substantial reduction in specific staining

• Correlation with Alternative Detection Methods:

  • Compare results with other validated SLC51A antibodies recognizing different epitopes

  • Correlate protein detection with mRNA levels using RT-PCR or RNA sequencing

• Western Blot Analysis:

  • Though not the primary application for this FITC-conjugated antibody, a parallel Western blot using a non-conjugated version of the same antibody can confirm specificity by molecular weight

  • Expected molecular weight for SLC51A: approximately 37-40 kDa

• Cross-reactivity Testing:

  • Test antibody reactivity against recombinant proteins with similar sequences

  • Particularly important when studying cells expressing both SLC51A and related transporters

What are common issues encountered when using FITC-conjugated antibodies and how can they be addressed?

When working with FITC-conjugated antibodies including the SLC51A antibody, researchers commonly encounter several technical challenges:

• Photobleaching:

  • Problem: FITC is particularly susceptible to photobleaching, leading to signal loss during imaging.

  • Solution: Minimize exposure to light during all experimental steps. Use anti-fade mounting media containing agents like p-phenylenediamine or commercial equivalents. Consider acquiring images of FITC channels first in multi-fluorophore experiments.

• Autofluorescence:

  • Problem: Biological samples often exhibit autofluorescence in the green spectrum, interfering with FITC signal detection.

  • Solution: Include unstained controls to assess background autofluorescence. Consider using spectral unmixing during image acquisition or treating samples with autofluorescence reducers like Sudan Black B (0.1-1%).

• pH Sensitivity:

  • Problem: FITC fluorescence is sensitive to pH, with optimal emission at pH 8.0 and significant reduction at lower pH.

  • Solution: Maintain consistent pH in buffers (ideally pH 7.4-8.0). For experiments involving acidic compartments, consider alternative fluorophores.

• Signal Intensity Issues:

  • Problem: Weak signal despite adequate protein expression.

  • Solution: Optimize antibody concentration through titration experiments. Ensure proper storage conditions are maintained (-20°C or -80°C, avoiding repeated freeze-thaw cycles) . Consider longer incubation times at 4°C.

• Non-specific Binding:

  • Problem: High background signal reducing detection specificity.

  • Solution: Increase blocking time/concentration (5-10% normal serum). Include 0.1-0.3% Triton X-100 in blocking buffer. Optimize antibody dilution, starting with the recommended range of 1:50-1:200 .

How can researchers optimize signal-to-noise ratio when using this antibody for fluorescence microscopy?

Optimizing signal-to-noise ratio is crucial for generating high-quality fluorescence microscopy data with the FITC-conjugated SLC51A antibody:

• Sample Preparation Optimization:

  • Fresh sample preparation with minimal storage time before fixation

  • Optimal fixation duration: typically 15-20 minutes with 4% PFA for membrane proteins

  • Complete quenching of fixative using glycine or ammonium chloride buffer

• Blocking Strategy Enhancement:

  • Extended blocking (1-2 hours at room temperature or overnight at 4°C)

  • Use combination blocking agents: 5% normal serum with 1-2% BSA

  • Addition of 0.1-0.3% Triton X-100 to blocking solution to reduce non-specific membrane binding

• Antibody Incubation Parameters:

  • Longer incubation at lower temperature (overnight at 4°C rather than 1-2 hours at room temperature)

  • Gentle agitation during incubation to ensure even antibody distribution

  • Thorough washing steps: at least 3-5 washes of 5-10 minutes each with PBS containing 0.1% Tween-20

• Microscopy Settings Adjustment:

  • Optimize exposure time to prevent oversaturation while capturing specific signals

  • Adjust gain and offset settings specific to the sample's signal intensity

  • Use appropriate filters optimized for FITC (excitation: ~490 nm, emission: ~520 nm)

  • Consider using confocal microscopy for improved signal-to-noise ratio through optical sectioning

• Post-acquisition Processing:

  • Background subtraction based on negative control samples

  • Deconvolution to improve signal clarity if appropriate for the microscopy system

  • Consistent application of processing parameters across all experimental conditions

What are appropriate storage conditions to maintain antibody performance over time?

Proper storage of the FITC-conjugated SLC51A antibody is essential for maintaining its performance characteristics over time:

• Temperature Requirements:

  • Store at -20°C or -80°C for long-term storage as specified by manufacturers

  • Avoid storing at 4°C for extended periods as this may lead to gradual loss of activity

• Aliquoting Strategy:

  • Upon receipt, divide the antibody into small single-use aliquots

  • Aliquot volumes should be sufficient for individual experiments to avoid repeated freeze-thaw cycles

  • Use sterile microcentrifuge tubes made of high-quality polypropylene

• Freeze-Thaw Considerations:

  • Minimize freeze-thaw cycles as they can significantly reduce antibody activity and FITC fluorescence intensity

  • If thawing is necessary, do so gradually at 4°C rather than at room temperature

  • Never heat the antibody to accelerate thawing

• Light Protection:

  • FITC is particularly sensitive to photobleaching

  • Store in amber or opaque tubes

  • Keep covered with aluminum foil during experimental procedures

  • Minimize exposure to light during all handling steps

• Buffer Conditions:

  • The antibody is supplied in a buffer containing 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as preservative

  • Do not dilute the stock antibody unless immediately using it

  • If dilution is necessary for an experiment, prepare fresh dilutions each time

• Documentation:

  • Maintain records of purchase date, lot number, and freeze-thaw cycles

  • Consider implementing a labeling system with dates of first thaw and number of uses

  • These records can help troubleshoot unexpected performance issues

How does FITC conjugation compare with other fluorophore conjugates for SLC51A detection?

When selecting between FITC and alternative fluorophore conjugates for SLC51A detection, researchers should consider the following comparative factors:

• Spectral Properties Comparison:

  Fluorophore	Excitation Max	Emission Max	Quantum Yield	Photostability	Typical Applications
  FITC	494 nm	520 nm	0.85	Low	IF, Flow Cytometry
  Alexa Fluor 488	496 nm	519 nm	0.92	High	IF, FACS, Confocal Microscopy
  PE (Phycoerythrin)	565 nm	575 nm	0.84	Medium	Flow Cytometry, More sensitive detection
  HRP	N/A (enzymatic)	N/A	N/A	N/A	ELISA, WB
  Biotin	N/A (non-fluorescent)	N/A	N/A	N/A	ELISA, Multi-step detection

• Advantages of FITC Conjugation:

  • Widely compatible with standard fluorescence filters and equipment

  • Relatively inexpensive compared to newer generation fluorophores

  • Small molecular size minimizes interference with antibody binding

  • Well-established protocols for use in multiple applications

• Limitations of FITC Compared to Alternatives:

  • More susceptible to photobleaching than Alexa Fluor dyes

  • Greater pH sensitivity than most alternative fluorophores

  • Lower signal-to-noise ratio in tissues with high autofluorescence

  • Less suitable for prolonged imaging or multi-day experiments

• Application-Specific Recommendations:

  • For fixed cell immunofluorescence: Alexa Fluor 488 may offer advantages over FITC

  • For flow cytometry of weakly expressed targets: PE conjugates might provide higher sensitivity

  • For multiplex experiments: Consider spectrally distinct fluorophores like Cy3 or Cy5

  • For specialized applications like FRET: Consider FITC paired with a compatible acceptor fluorophore

• Cost-Benefit Considerations:

  • FITC conjugates are typically more affordable than newer generation fluorophores

  • For routine detection of strongly expressed targets, FITC is often sufficient

  • For challenging applications or weakly expressed proteins, investment in alternative conjugates may be justified

What emerging techniques could enhance the study of SLC51A expression and function?

Several innovative methodologies offer promising approaches for advancing SLC51A research beyond conventional antibody applications:

• CRISPR-Cas9 Genome Editing:

  • Generation of endogenous fluorescent protein tags fused to SLC51A to monitor expression without antibodies

  • Creation of conditional knockout models to study tissue-specific functions

  • Introduction of specific point mutations to investigate structure-function relationships

• Super-Resolution Microscopy:

  • Techniques like STORM, PALM, or STED microscopy can reveal SLC51A distribution at nanoscale resolution

  • Co-localization studies with SLC51B at unprecedented detail

  • Visualization of membrane microdomains where transporters function

• Live-Cell Imaging Approaches:

  • Development of cell lines expressing SLC51A tagged with photostable fluorescent proteins

  • Real-time monitoring of transporter trafficking in response to physiological stimuli

  • FRAP (Fluorescence Recovery After Photobleaching) to study mobility and turnover rates

• Single-Cell Analysis:

  • Single-cell RNA sequencing to correlate SLC51A expression with other genes across heterogeneous cell populations

  • Mass cytometry (CyTOF) for simultaneous detection of dozens of proteins alongside SLC51A

  • Spatial transcriptomics to map SLC51A expression patterns within intact tissues

• Organoid and Microphysiological Systems:

  • Investigation of SLC51A function in intestinal or liver organoids

  • Development of "gut-on-a-chip" models to study enterohepatic circulation

  • Patient-derived organoids to investigate disease-specific alterations

• Computational Approaches:

  • Molecular dynamics simulations of SLC51A/SLC51B interactions

  • Machine learning algorithms to predict transporter substrate specificity

  • Systems biology approaches to integrate SLC51A into broader metabolic networks

How can researchers integrate SLC51A antibody-based studies with functional transport assays?

Integrating structural detection of SLC51A using antibodies with functional assays provides a more comprehensive understanding of transporter biology:

• Correlation Analysis Framework:

  • Quantify SLC51A expression levels using the FITC-conjugated antibody via flow cytometry or quantitative immunofluorescence

  • In parallel, measure transport activity of labeled bile acids or other substrates

  • Perform regression analysis to determine relationship between expression and function

  • Identify potential threshold effects or non-linear relationships

• Sequential Experimental Design:

  • First, confirm SLC51A expression and localization using immunofluorescence

  • Next, conduct transport assays with radiolabeled or fluorescently-labeled substrates

  • Finally, manipulate expression levels (via siRNA or overexpression) and reassess both parameters

  • This approach establishes causality between expression and function

• Co-localization with Functional Readouts:

  • Combine SLC51A immunostaining with fluorescent bile acid analogs

  • Live-cell imaging to track both protein localization and substrate movement

  • Analysis of transporter clustering in relation to transport efficiency

• Integrated Multi-Parameter Analysis:

  • Develop cell models with fluorescent reporters for both SLC51A expression and substrate transport

  • High-content imaging to simultaneously assess multiple parameters

  • Machine learning algorithms to identify patterns and correlations across large datasets

• Complementary Technique Selection:

  Antibody Application	Complementary Functional Assay	Integrated Analysis Approach
  Flow cytometry	Radiolabeled substrate uptake	Correlation of MFI with transport rate per cell
  Immunofluorescence	Fluorescent substrate imaging	Co-localization analysis at membrane domains
  Western blotting	Membrane vesicle transport	Correlation of expression with Vmax values
  IP-MS	Substrate spectrum analysis	Linking protein interactions to transport preferences