SLC22A25 Antibody, FITC conjugated

What is SLC22A25 and what role does it play in cellular transport mechanisms?

SLC22A25 belongs to the solute carrier family 22, a group of membrane transport proteins responsible for mediating the transport of organic ions across cell membranes. This protein functions as an integral component of the plasma membrane with multiple transport capabilities including inorganic anion exchanger activity, sodium-independent organic anion transmembrane transporter activity, and urate transmembrane transporter activity . These transport functions are critical for cellular homeostasis and the movement of endogenous compounds, drugs, and toxins across cellular membranes. SLC22A25 is specifically classified as an organic anion transporter (also known as UST6), positioning it within a broader family of transporters that play significant roles in drug disposition and elimination . Understanding SLC22A25's function is particularly important for research in pharmacokinetics, drug metabolism, and the study of genetic variations that may affect drug efficacy or toxicity.

What are the key specifications of the FITC-conjugated SLC22A25 antibody?

The FITC-conjugated SLC22A25 antibody (AA 31-145) is a polyclonal antibody generated in rabbits that specifically targets amino acids 31-145 of the human SLC22A25 protein . This antibody has been purified using Protein G affinity chromatography to achieve >95% purity and is provided in liquid form with a preservative (0.03% Proclin 300) in a buffer containing 50% glycerol and 0.01M PBS at pH 7.4 . The antibody is directly conjugated to FITC (Fluorescein Isothiocyanate), a fluorescent dye with excitation maximum at approximately 495 nm and emission maximum around 519 nm, making it compatible with standard fluorescence detection systems . While specific applications may require validation, related SLC22A25 antibodies have been successfully used in applications including ELISA (recommended dilution 1:2000-1:10000), Western blot (1:500-1:5000), immunohistochemistry (1:100-1:300), and immunofluorescence (1:50-1:200) . The antibody demonstrates specific reactivity with human SLC22A25 and has been tested in cell lines including HeLa .

How does the epitope specificity (AA 31-145) affect antibody performance?

The epitope specificity of this SLC22A25 antibody, targeting amino acids 31-145, has significant implications for its research applications and performance characteristics. This specific region represents approximately 18% of the full SLC22A25 protein (assuming a typical transporter size of ~550-650 amino acids) and likely encompasses unique sequence elements that distinguish it from other related transporters . The relatively large epitope region (115 amino acids) suggests this antibody may recognize multiple epitopes within this segment, potentially providing robust detection even if some epitopes are partially masked or altered in different experimental conditions . This specific epitope region may correspond to an extracellular or cytoplasmic domain of the transporter, which would affect accessibility in different applications (non-permeabilized vs. permeabilized conditions) . The immunogen used to generate this antibody was a recombinant human SLC22A25 protein fragment containing amino acids 31-145, which influences both the specificity and the applications for which this antibody is most suitable .

What advantages does a polyclonal FITC-conjugated antibody offer for SLC22A25 research?

Polyclonal antibodies like this FITC-conjugated SLC22A25 antibody offer several distinct advantages for transporter research. The polyclonal nature enables recognition of multiple epitopes within the target region (AA 31-145), potentially providing stronger signal intensity as multiple antibodies can bind to each SLC22A25 molecule . This characteristic makes the antibody particularly valuable for detecting low-abundance membrane proteins like transporters. Additionally, polyclonal antibodies tend to be more tolerant of minor changes in protein conformation or denaturation, maintaining reactivity across various experimental conditions including fixed tissues, denatured proteins in Western blots, and native proteins in flow cytometry . The direct FITC conjugation eliminates the need for secondary antibodies, reducing background from secondary antibody cross-reactivity while simplifying and shortening experimental protocols . Furthermore, this combination makes the antibody exceptionally well-suited for multicolor immunofluorescence studies when paired with antibodies conjugated to spectrally distinct fluorophores that can help elucidate SLC22A25's relationship with other cellular components.

What is known about SLC22A25 expression patterns in human tissues?

While comprehensive expression data specific to SLC22A25 is somewhat limited in the available literature, some key patterns can be identified. Based on related studies of the SLC22 family, SLC22A25 likely follows expression patterns similar to other organic anion transporters, with predominant expression in tissues involved in drug disposition and elimination . Validation data from related SLC22A25 antibodies shows successful detection in HeLa whole cell lysates, indicating expression in this cervical cancer cell line . As a membrane transporter involved in organic anion and urate transport, SLC22A25 would be expected to show highest expression in epithelial barriers and excretory organs such as kidney tubules, hepatocytes in the liver, and enterocytes in the intestine . The specific FITC-conjugated antibody targeting amino acids 31-145 can be valuable for mapping precise expression patterns across tissues and cell types through techniques like immunohistochemistry and flow cytometry . Researchers interested in detailed expression profiling should consider using this antibody in conjunction with qPCR or RNA-seq approaches to correlate protein detection with transcript levels across diverse tissue panels.

How should I optimize immunofluorescence protocols for SLC22A25 detection with this FITC-conjugated antibody?

Optimizing immunofluorescence protocols for the FITC-conjugated SLC22A25 antibody requires careful consideration of several parameters. Begin with fixation optimization, testing both 4% paraformaldehyde (10-15 minutes at room temperature) and milder 2% PFA fixation protocols, as membrane transporters like SLC22A25 can be sensitive to overfixation that might mask epitopes . Permeabilization should be tested with a titration of detergent concentrations (e.g., 0.1-0.3% Triton X-100 or 0.01-0.1% saponin), with the latter often being gentler for membrane proteins . For blocking, use 5-10% normal serum (from a species different from the antibody host) with 1-2% BSA to minimize background while maintaining specific signal detection . Antibody concentration should be optimized through titration experiments starting at the recommended 1:50 dilution for immunofluorescence and adjusting based on signal-to-background ratio . Consider comparing both room temperature incubation (1-2 hours) and 4°C overnight incubation to determine optimal binding conditions for your specific samples. After staining, mount slides with anti-fade mounting media containing DAPI for nuclear counterstaining, and seal with nail polish or commercial sealants to prevent drying and oxidation that can compromise FITC fluorescence.

What controls are essential when using this SLC22A25 antibody in experimental studies?

Implementing comprehensive controls is critical for generating reliable data with the FITC-conjugated SLC22A25 antibody. For negative controls, include an isotype control using FITC-conjugated rabbit IgG at matching concentration to assess non-specific binding from the antibody class, a no-primary antibody control to evaluate autofluorescence and secondary antibody background (if using additional detection systems), and cells/tissues known to lack SLC22A25 expression as biological negative controls . Positive controls should include cell lines with confirmed SLC22A25 expression (such as HeLa based on validation data) or recombinant expression systems overexpressing the target protein . Specificity controls should include pre-absorption with the immunizing peptide (AA 31-145) when possible, or comparison with another SLC22A25 antibody targeting a different epitope region to confirm staining patterns . For functional validation, consider correlating antibody staining patterns with SLC22A25 knockdown experiments using siRNA or CRISPR approaches, which should reduce signal proportionally to knockdown efficiency if the antibody is specific . Additionally, when performing multicolor experiments, include single-color controls to assess spectral bleed-through and establish proper compensation parameters for accurate signal separation.

How can I achieve optimal signal-to-noise ratio in flow cytometry with this antibody?

Achieving optimal signal-to-noise ratio in flow cytometry with the FITC-conjugated SLC22A25 antibody requires systematic optimization of multiple parameters. Start with cell preparation, ensuring high viability (>90%) and single-cell suspensions while avoiding harsh enzymatic dissociation methods that might damage membrane proteins like SLC22A25 . For antibody concentrations, begin with approximately 1 μg per million cells and perform titration experiments to identify the concentration that maximizes the separation between positive and negative populations . Buffer optimization is crucial - use a flow cytometry buffer containing 1-2% protein (BSA or FBS) and 0.1% sodium azide, with pH maintained between 7.2-7.4 to preserve FITC fluorescence properties . For membrane transporters like SLC22A25, compare staining of live cells versus fixed cells, as some epitopes may be better preserved in one condition over the other . When analyzing data, establish proper gating strategies using isotype controls and unstained samples to accurately distinguish positive populations from background . For multiparameter analysis, include fluorescence-minus-one (FMO) controls to set boundaries between positive and negative populations, and compensate for FITC spillover into other channels using single-color controls .

What approaches should I use to validate antibody specificity in my experimental system?

Validating specificity of the FITC-conjugated SLC22A25 antibody in your experimental system requires a multi-faceted approach. Begin with genetic validation by performing siRNA or shRNA knockdown of SLC22A25, which should proportionally reduce antibody staining if specific, or CRISPR/Cas9 knockout which should eliminate specific signal entirely . Complementary to knockdown, overexpression of SLC22A25 in low/non-expressing cells should increase signal intensity in a dose-dependent manner . Biochemical validation should include Western blot analysis (with a non-conjugated version of the antibody) to confirm detection of a band at the expected molecular weight (~62 kDa) . Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide (AA 31-145) before staining, should substantially reduce or eliminate specific signal . For cross-reactivity assessment, perform sequence alignment analysis of the epitope region (AA 31-145) with other SLC family members to identify potential cross-reactive proteins, then test the antibody against cells expressing those related transporters . Correlative validation comparing antibody staining patterns with mRNA expression levels across different cell types can provide additional evidence of specificity. Finally, consider method validation by comparing results from this FITC-conjugated antibody with those obtained using alternative SLC22A25 antibodies targeting different epitopes or using different detection methods.

How can I incorporate this antibody into multiplex immunofluorescence studies?

Incorporating the FITC-conjugated SLC22A25 antibody into multiplex immunofluorescence studies requires careful panel design and optimization. Start by selecting complementary fluorophores with minimal spectral overlap with FITC (excitation ~495 nm, emission ~519 nm), such as DAPI for nuclei, Cy3/Alexa Fluor 555 for red channel, and Cy5/Alexa Fluor 647 for far-red channel . For antibody panel design, consider host species compatibility - ideally using antibodies raised in different host species to avoid cross-reactivity between secondary detection systems for other targets . When designing the staining protocol, determine the optimal staining sequence through experimentation; generally start with the FITC-conjugated SLC22A25 antibody since directly conjugated antibodies often show reduced signal compared to amplified detection methods used for other targets . Include extensive washing steps between different antibodies to minimize cross-talk and background. For image acquisition, use sequential scanning on confocal microscopes to prevent spectral bleed-through, and consistently apply appropriate exposure settings across all experimental conditions . For quantitative analysis, use software capable of spectral unmixing if needed, and analyze co-localization using established methods such as Pearson's correlation coefficient or Manders' overlap coefficient. Always include single-stained controls for each fluorophore to set proper acquisition parameters and facilitate post-acquisition spectral unmixing if required.

How can I use this antibody to study SLC22A25 trafficking and membrane localization?

Studying SLC22A25 trafficking and membrane localization with this FITC-conjugated antibody requires sophisticated experimental approaches. Begin with pulse-chase experiments, where surface SLC22A25 is labeled using the antibody in non-permeabilized conditions, followed by temperature shifts or endocytic triggers to initiate internalization; subsequent fixation, permeabilization, and staining with markers for different endocytic compartments can reveal trafficking pathways . For dynamic live-cell imaging, the FITC-conjugated antibody can be used to label surface SLC22A25 in live cells at 4°C (to prevent internalization), followed by warming to 37°C and time-lapse imaging to track internalization and recycling in real-time, though photobleaching considerations are important with FITC . To assess steady-state localization, co-staining with markers for plasma membrane (Na+/K+-ATPase), early endosomes (EEA1), recycling endosomes (Rab11), late endosomes (Rab7), and lysosomes (LAMP1) using spectrally distinct fluorophores can reveal the distribution of SLC22A25 across these compartments . For regulation studies, treatments with stimuli known to modulate transporter trafficking (e.g., protein kinase activators, endocytic modulators) followed by quantification of surface versus internal SLC22A25 can elucidate regulatory mechanisms . Higher-resolution studies using techniques like TIRF microscopy can reveal SLC22A25 organization within the plasma membrane, potentially identifying specialized membrane domains where the transporter concentrates .

What strategies can distinguish between SLC22A25 and other closely related transporters?

Distinguishing SLC22A25 from other closely related transporters requires strategic approaches to overcome potential cross-reactivity. Begin with computational analysis by performing sequence alignment of the antibody's target region (AA 31-145) with corresponding regions in related SLC22 family members to identify unique versus conserved epitopes; this information can predict potential cross-reactivity and guide validation experiments . For experimental validation, generate a panel of cells expressing individual SLC transporters (either through stable transfection or inducible expression systems) and test the antibody against each to identify any cross-reactive family members . Knockdown validation using siRNA libraries targeting individual SLC transporters can help confirm antibody specificity - only knockdown of SLC22A25 should reduce specific staining if the antibody is truly selective . Co-staining approaches using the FITC-conjugated SLC22A25 antibody alongside antibodies against related transporters (labeled with spectrally distinct fluorophores) can reveal whether the staining patterns are distinct or overlapping . For functional discrimination, correlate antibody staining with transport assays specific for different SLC22 family members; cells showing SLC22A25 antibody staining should demonstrate transport properties consistent with SLC22A25 rather than related transporters . Advanced methods like proximity ligation assays using the SLC22A25 antibody paired with antibodies against suspected cross-reactive targets can quantitatively assess the degree of potential cross-reactivity in situ.

How might this antibody be utilized in studies of drug transport and pharmacokinetics?

This FITC-conjugated SLC22A25 antibody offers valuable applications for drug transport and pharmacokinetic studies. For transporter expression profiling, use the antibody to screen cell lines, primary tissues, and patient samples to correlate SLC22A25 expression levels with drug response or clearance rates, potentially identifying expression-based biomarkers for drug disposition . In drug-transporter interaction studies, assess whether drug candidates alter SLC22A25 trafficking or expression through quantitative immunofluorescence before and after drug exposure, providing insights into potential drug-drug interactions at the transporter level . For mechanistic studies, combine immunolocalization of SLC22A25 using this antibody with fluorescent drug analogs to visualize co-localization patterns and transport kinetics, potentially using live-cell imaging approaches if photobleaching can be controlled . Structure-function studies can utilize site-directed mutagenesis of SLC22A25 followed by antibody detection to determine how specific mutations affect transporter expression, localization, and stability, correlating these parameters with transport function . For translational applications, the antibody can help validate in vitro findings in more complex systems such as tissue slices, organoids, or xenograft models, providing visualization of transporter expression in physiologically relevant contexts . Inter-individual variation studies can utilize the antibody to assess how genetic polymorphisms in SLC22A25 impact protein expression and localization, potentially explaining pharmacokinetic differences between individuals .

What are the considerations for using this antibody in frozen versus fixed tissue sections?

Using the FITC-conjugated SLC22A25 antibody with frozen versus fixed tissue sections presents distinct considerations that impact experimental outcomes. For frozen sections, tissue preservation is crucial - snap freezing in OCT compound followed by cryosectioning at -20°C to -25°C helps maintain antigenic epitopes and cellular architecture while preserving the membrane localization of transporters like SLC22A25 . Post-sectioning fixation should be gentle, with brief exposure (5-10 minutes) to 2-4% paraformaldehyde to minimize epitope masking while providing sufficient structural stability . For formalin-fixed paraffin-embedded (FFPE) tissues, antigen retrieval becomes essential - test both heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0) and enzymatic retrieval methods (proteinase K or trypsin) to determine which best recovers SLC22A25 epitopes while maintaining tissue morphology . Autofluorescence management differs between preparations - frozen sections typically have lower autofluorescence but may require brief treatment with 0.1% sodium borohydride, while FFPE sections often benefit from more aggressive treatments such as Sudan Black B (0.1-0.3%) or specialized commercial autofluorescence quenchers . Antibody concentration optimization usually requires higher concentrations for FFPE sections (start at 1:50 dilution) compared to frozen sections (start at 1:100 dilution) due to different degrees of epitope availability . For quantitative comparisons between samples, it's critical to maintain consistent processing, fixation, and staining protocols across all specimens to ensure comparable SLC22A25 detection.

How can this antibody contribute to understanding SLC22A25 regulation under pathophysiological conditions?

This FITC-conjugated SLC22A25 antibody can significantly advance our understanding of transporter regulation under pathophysiological conditions through multiple research approaches. For disease-state comparisons, quantitative immunofluorescence can assess SLC22A25 expression and localization changes in tissues from normal versus disease states (e.g., renal or hepatic disease, inflammatory conditions), potentially revealing how pathology impacts this transporter . Regulatory mechanism studies can combine the antibody with treatments mimicking pathophysiological conditions (hypoxia, inflammation, oxidative stress) to visualize acute changes in SLC22A25 trafficking, internalization, or degradation in response to cellular stress . Drug response studies in disease models can utilize the antibody to determine whether therapeutic interventions restore normal SLC22A25 expression and localization patterns, correlating these changes with recovery of transporter function . For mechanistic investigations, co-localization studies combining the FITC-conjugated SLC22A25 antibody with markers for regulatory proteins (kinases, ubiquitin ligases, trafficking regulators) using spectrally distinct fluorophores can reveal protein-protein interactions that regulate the transporter under different conditions . Translational research applications include using the antibody to stratify patient samples based on SLC22A25 expression/localization patterns, potentially identifying patient subgroups with altered drug transport capacity that might benefit from personalized dosing strategies . For longitudinal studies, repeated sampling and staining for SLC22A25 during disease progression or therapeutic intervention can track dynamic changes in transporter regulation over time, providing insights into adaptive responses and potential compensatory mechanisms.