TAP2 Antibody, FITC conjugated

What is TAP2 and why is it an important research target?

TAP2 is a critical component of the antigen processing machinery that works in conjunction with TAP1 to form a functional heterodimeric transporter. This complex mediates the unidirectional translocation of peptide antigens from the cytosol to the endoplasmic reticulum (ER) for loading onto MHC class I molecules, a crucial step in the cellular immune response pathway . TAP2 functions by utilizing the chemical energy of ATP to export peptides against their concentration gradient, alternating between "inward-facing" and "outward-facing" conformational states during the transport cycle . The protein is essential for proper antigen presentation and immune surveillance, making it a significant target in immunology research, particularly in studies of viral immune evasion, autoimmunity, and cancer immunotherapy . TAP2 dysfunction has been implicated in various immunological disorders, highlighting its importance as a research subject.

What are the key features of TAP2 Antibody, FITC conjugated products?

TAP2 Antibody, FITC conjugated (such as ABIN7144331) typically consists of a polyclonal antibody raised in rabbits against a specific epitope region (often amino acids 473-615) of human TAP2 protein . The fluorescein isothiocyanate (FITC) conjugation enables direct fluorescent detection without the need for secondary antibodies, streamlining immunofluorescence protocols . These antibodies undergo protein G purification to achieve >95% purity and are often formulated in buffers containing preservatives like Proclin 300 and glycerol to maintain stability . Most commercially available versions demonstrate reactivity primarily with human TAP2, though some may cross-react with mouse or rat orthologs depending on the specific epitope targeted . Their polyclonal nature provides robust signal detection by recognizing multiple epitopes within the targeted region of the TAP2 protein.

What applications are TAP2 Antibody, FITC conjugated suitable for?

The FITC-conjugated TAP2 antibodies are optimized for several research applications:

While the exact applications may vary by manufacturer, these antibodies are particularly valuable for multicolor flow cytometry and immunofluorescence studies where direct labeling simplifies experimental design .

What is the optimal protocol for using TAP2 Antibody, FITC conjugated in flow cytometry?

For intracellular flow cytometry with TAP2 Antibody, FITC conjugated, follow this optimized protocol:

• Cell preparation: Harvest cells (1-5×10⁶) and wash twice with ice-cold PBS containing 1% BSA.

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature to preserve cellular architecture.

• Permeabilization: Treat cells with 0.1% saponin or commercial permeabilization buffer for 15 minutes to allow antibody access to intracellular TAP2.

• Blocking: Incubate cells with 10% normal serum in permeabilization buffer for 30 minutes to reduce non-specific binding.

• Primary antibody staining: Add TAP2 Antibody, FITC conjugated at an optimized dilution (typically 1:50 to 1:200) and incubate for 45-60 minutes at room temperature in the dark to prevent photobleaching .

• Washing: Perform 3 washes with permeabilization buffer to remove unbound antibody.

• Analysis: Analyze samples immediately or fix additionally with 1% paraformaldehyde for later analysis, storing protected from light at 4°C.

For optimal results, always include appropriate controls: an isotype control conjugated to FITC, unstained cells, and positive control cells known to express TAP2 (such as B-lymphoblastoid cell lines or specific tumor cell lines like MCF-7) .

How should TAP2 Antibody, FITC conjugated be stored and handled to maintain optimal performance?

To maintain the integrity and performance of TAP2 Antibody, FITC conjugated:

• Storage temperature: Store at -20°C for long-term storage and 4°C for short-term use (less than one month) .

• Protection from light: Always keep FITC-conjugated antibodies protected from light during storage and handling to prevent photobleaching of the fluorophore.

• Aliquoting: Upon receipt, divide the antibody into small, single-use aliquots to avoid repeated freeze-thaw cycles, which can degrade both the antibody and the FITC conjugate.

• Buffer conditions: The antibody is typically supplied in a stabilizing buffer containing glycerol (often 50%) and preservatives such as 0.03% Proclin 300 in PBS at pH 7.4 . Maintain these conditions when diluting.

• Centrifugation: Briefly centrifuge the antibody vial before opening to collect liquid at the bottom of the tube.

• Handling: When pipetting, avoid introducing bubbles which can lead to protein denaturation at the air-liquid interface.

• Contamination prevention: Always use clean pipette tips and sterile techniques to prevent microbial contamination.

Following these guidelines will help maintain antibody activity and fluorescence intensity throughout the shelf life of the product, typically 12 months from date of receipt when properly stored .

How can TAP2 Antibody, FITC conjugated be used to investigate TAP2 dysregulation in cancer models?

TAP2 dysregulation is a significant mechanism by which cancer cells evade immune surveillance. To investigate this phenomenon using TAP2 Antibody, FITC conjugated:

• Comparative expression analysis: Utilize flow cytometry with TAP2 Antibody, FITC conjugated to quantitatively compare TAP2 expression levels between cancer cell lines and corresponding normal tissues. Calculate mean fluorescence intensity (MFI) ratios to determine relative expression changes .

• Immunofluorescence co-localization studies: Perform dual labeling with TAP2 Antibody, FITC conjugated and markers for the endoplasmic reticulum (such as calnexin or PDI) to assess whether TAP2 localization is altered in cancer cells, which might indicate functional defects even when expression is maintained.

• Tumor microenvironment analysis: Analyze TAP2 expression in tumor-infiltrating immune cells versus peripheral blood counterparts to understand how the tumor microenvironment might affect antigen presentation machinery.

• Treatment response monitoring: Use TAP2 Antibody, FITC conjugated to track changes in TAP2 expression following treatment with immunomodulatory agents or chemotherapeutics that might restore antigen presentation.

• Genetic manipulation validation: Confirm successful TAP2 knockdown or overexpression in mechanistic studies through flow cytometry or microscopy with the FITC-conjugated antibody.

Research has shown that TAP2 downregulation correlates with disease progression and poor prognosis in multiple cancer types, including ovarian cancer, colorectal carcinoma, and melanoma . Using FITC-conjugated antibodies allows for precise quantification of these expression changes, potentially identifying patients who might benefit from immunotherapy approaches targeting antigen presentation restoration.

What are effective troubleshooting strategies for weak or non-specific signals when using TAP2 Antibody, FITC conjugated?

When encountering signal issues with TAP2 Antibody, FITC conjugated, implement these methodological solutions:

For immunohistochemistry applications specifically, optimizing antigen retrieval is critical - test both heat-induced epitope retrieval with citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0) to determine which better exposes the TAP2 epitope .

How does the amino acid region 473-615 of TAP2 relate to its functional domains and antibody recognition?

The amino acid region 473-615 of TAP2 encompasses functionally significant domains that influence both protein activity and antibody recognition:

• Structural significance: This region contains portions of the nucleotide-binding domain (NBD) of TAP2, which is responsible for ATP binding and hydrolysis, providing the energy needed for peptide transport . The region contributes to the conformational changes that occur during the transport cycle, alternating between "inward-facing" and "outward-facing" states.

• Conformational epitopes: Antibodies targeting this region may recognize conformational epitopes that are sensitive to the protein's folding state. This makes proper sample preparation crucial for preserving these structural elements during immunodetection.

• Conservation considerations: This region shows moderate conservation across species, which explains why some TAP2 antibodies targeting this epitope may cross-react with human samples but not with mouse or rat orthologs .

• Functional relevance: Amino acids in this region contribute to the peptide translocation pathway and may be directly involved in the binding of viral immune evasion proteins that target TAP2, such as herpes simplex virus ICP47 or human cytomegalovirus US6 glycoprotein .

• Post-translational modifications: This region contains potential phosphorylation sites that may regulate TAP2 activity and could affect antibody binding depending on the phosphorylation state.

When using antibodies targeting this region, researchers should consider that different functional states of TAP2 (ATP-bound, peptide-bound, etc.) might affect epitope accessibility and antibody recognition efficiency. This is particularly important when studying TAP2 in different cellular contexts or under conditions that might alter its conformational state .

What are the essential controls needed when working with TAP2 Antibody, FITC conjugated?

To ensure reliable and interpretable results with TAP2 Antibody, FITC conjugated, implement these essential controls:

• Isotype control: Include a FITC-conjugated rabbit IgG isotype control at the same concentration as the TAP2 antibody to assess non-specific binding and set appropriate gates in flow cytometry or background thresholds in microscopy .

• Positive cellular control: Use cell lines known to express TAP2 consistently, such as:

  • MCF-7 (human breast cancer cells)

  • SH-SY5Y (human neuroblastoma cells)

  • B-lymphoblastoid cell lines

  • Interferon-γ stimulated cells (TAP2 is upregulated by IFN-γ)

• Negative cellular control: Include cell lines with low or absent TAP2 expression, such as certain TAP-deficient cell lines or cells where TAP2 has been knocked down using siRNA/shRNA.

• Blocking/competition control: Pre-incubate the antibody with recombinant TAP2 protein (particularly the immunogen fragment, aa 473-615) before staining to confirm binding specificity .

• Unstained control: Include completely unstained samples to establish baseline autofluorescence.

• Single-color controls: For multicolor experiments, prepare single-color controls to establish compensation settings.

• Secondary-only control: For indirect immunofluorescence experiments, include a control with only the secondary reagent to assess non-specific binding.

• Biological treatment control: When studying TAP2 modulation, include appropriate treatment controls (e.g., IFN-γ treatment should increase TAP2 expression).

Documenting and reporting these controls in publications is essential for result validation and experimental reproducibility in TAP2 research.

How can TAP2 Antibody, FITC conjugated be used effectively in multiplex immunofluorescence studies?

For successful multiplex immunofluorescence studies incorporating TAP2 Antibody, FITC conjugated:

• Fluorophore selection and panel design:

  • Since FITC emits in the green spectrum (peak emission ~519nm), pair with fluorophores that have minimal spectral overlap such as DAPI (blue), Cy3/PE (orange/yellow), Cy5/APC (far red).

  • For flow cytometry panels, place FITC in a channel detecting markers of intermediate expression level due to its moderate brightness compared to newer fluorophores.

• Staining sequence optimization:

  • For co-localization studies with TAP1, MHC Class I, or other components of the peptide loading complex, stain with TAP2 Antibody, FITC conjugated first, followed by other markers.

  • When combining with antibodies from the same host species (rabbit), employ sequential staining with complete blocking steps between antibodies.

• Imaging considerations:

  • Use narrow bandpass filters to minimize bleed-through between channels.

  • Acquire single-stained controls for each fluorophore to establish proper compensation/unmixing parameters.

  • Consider photobleaching characteristics of FITC when designing acquisition sequences (image FITC channels early in the sequence).

• Sample preparation refinements:

  • For tissue sections, perform tyramide signal amplification (TSA) to enhance FITC signal when needed.

  • Test alternative fixatives beyond paraformaldehyde (such as methanol or glyoxal) if certain epitopes show poor detection.

• Data analysis approach:

  • Employ computational analysis tools like CellProfiler or QuPath for automated quantification of co-localization.

  • Calculate Pearson's correlation coefficient or Manders' overlap coefficient to quantify the degree of co-localization between TAP2 and other proteins of interest.

A particularly informative multiplexing approach involves combining TAP2 Antibody, FITC conjugated with antibodies against TAP1, tapasin, MHC-I, and ER markers to comprehensively analyze the entire peptide loading complex in various experimental conditions .

How can TAP2 Antibody, FITC conjugated be used to study the impact of viral immune evasion strategies?

Viral immune evasion frequently targets the TAP complex to prevent antigen presentation. TAP2 Antibody, FITC conjugated can be employed to study these mechanisms through:

• Viral protein-TAP2 interaction studies:

  • Perform co-immunoprecipitation followed by immunoblotting to identify interactions between viral proteins and TAP2.

  • Use immunofluorescence microscopy with TAP2 Antibody, FITC conjugated to visualize changes in TAP2 localization upon viral protein expression.

  • Employ FRET (Fluorescence Resonance Energy Transfer) techniques with TAP2 Antibody, FITC conjugated and differently labeled viral proteins to detect direct interactions.

• Quantitative expression analysis:

  • Use flow cytometry with TAP2 Antibody, FITC conjugated to measure changes in TAP2 expression levels during viral infection timecourses.

  • Compare expression in infected versus uninfected cells within the same sample to control for technical variables.

• Functional correlation studies:

  • Correlate TAP2 expression (measured by FITC intensity) with surface MHC-I levels (using differently conjugated MHC-I antibodies) to establish functional consequences.

  • Perform peptide transport assays in parallel with TAP2 expression analysis to correlate expression with function.

• Rescue experiments:

  • After documenting TAP2 downregulation or mislocalization during viral infection, attempt rescue through treatment with proteasome inhibitors, interferon-γ, or viral protein inhibitors.

  • Monitor restoration of normal TAP2 patterns using the FITC-conjugated antibody.

Several viruses employ specific TAP inhibition strategies: herpes simplex virus ICP47 blocks the peptide-binding site, human cytomegalovirus US6 inhibits ATP binding to TAP1, and human adenovirus E3-19K prevents MHC class I/TAP association . These mechanisms can be specifically investigated using TAP2 Antibody, FITC conjugated to visualize their effects on TAP complex integrity and localization.

What quantitative approaches can be used with TAP2 Antibody, FITC conjugated for comparative expression studies?

To perform rigorous quantitative analysis of TAP2 expression using FITC-conjugated antibodies:

• Flow cytometry quantification methods:

  • Mean/Median Fluorescence Intensity (MFI): Calculate the average FITC signal intensity within the positive population, normalizing to isotype controls.

  • Molecules of Equivalent Soluble Fluorochrome (MESF): Convert arbitrary fluorescence units to absolute values using calibration beads with defined FITC molecules.

  • Quantitative Flow Cytometry (QFCM): Employ calibration standards to determine the antibody binding capacity (ABC) and convert to absolute receptor numbers.

• Image-based quantification approaches:

  • Integrated Density Measurement: Calculate the product of area and mean signal intensity in defined cellular compartments.

  • Colocalization Quantification: Determine Pearson's or Manders' coefficients to quantify TAP2 association with other components of the antigen processing machinery.

  • Single-Molecule Detection: For super-resolution microscopy, count individual fluorescent spots to estimate molecule numbers.

• Standardization practices:

  • Use consistent photomultiplier tube (PMT) voltage settings across experiments.

  • Include calibrated beads in each experiment to normalize for day-to-day instrument variations.

  • Perform antibody titration experiments to ensure measurements are made in the linear range of detection.

• Comparative analysis methods:

  • Fold-change calculations: Express results as fold-change relative to appropriate controls rather than absolute values.

  • Standard curve generation: Create cellular standards with known TAP2 expression levels through genetic manipulation for relative quantification.

  • Western blot correlation: Validate flow cytometry or microscopy quantification with parallel Western blot analysis when possible.

This quantitative approach has been successfully applied to demonstrate differential TAP2 expression across tissues and its modulation during immune responses, providing insights into the regulation of antigen presentation in various physiological and pathological contexts .

How can researchers optimize TAP2 Antibody, FITC conjugated for use in tissue microarray (TMA) analysis?

For effective use of TAP2 Antibody, FITC conjugated in tissue microarray (TMA) analysis:

• Tissue preparation considerations:

  • Fixation optimization: Test parallel TMAs with different fixation times (6-24 hours) to determine optimal epitope preservation for TAP2.

  • Antigen retrieval methods: Compare heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) versus Tris-EDTA buffer (pH 9.0) to maximize signal while maintaining tissue morphology .

  • Section thickness: Standardize to 4-5μm sections for optimal antibody penetration and signal intensity.

• Staining protocol refinements:

  • Signal amplification: Consider tyramide signal amplification (TSA) to enhance FITC signal, especially for tissues with lower TAP2 expression.

  • Background reduction: Apply Sudan Black B (0.1-0.3%) treatment to reduce tissue autofluorescence, particularly important with FITC detection.

  • Automated platforms: Validate staining consistency across the TMA using automated staining platforms with precise timing and washing controls.

• Analysis approaches:

  • Digital pathology integration: Utilize whole slide imaging scanners with appropriate FITC filter sets for consistent image acquisition.

  • Machine learning algorithms: Develop training sets for automated detection of TAP2-positive cells versus background in complex tissue architectures.

  • Multi-parameter scoring: Establish a semi-quantitative scoring system (0-3+) for TAP2 expression that accounts for both intensity and percentage of positive cells.

• Validation strategies:

  • Include control cores: Incorporate known TAP2-positive tissues (lymphoid tissues, specific tumor types) and TAP2-negative tissues in each TMA.

  • Technical replicates: Include at least duplicate cores from each case to account for tissue heterogeneity.

  • Correlation analysis: Perform parallel assessment with conventional IHC using HRP-conjugated TAP2 antibodies for method validation .

This approach enables high-throughput analysis of TAP2 expression across large cohorts of patient samples, facilitating clinicopathological correlations and potential biomarker development for immunotherapy response prediction .

What are the advantages and limitations of TAP2 Antibody, FITC conjugated compared to other detection methods?

Understanding the comparative profile of FITC-conjugated TAP2 antibodies helps researchers select the optimal detection tool:

How can TAP2 Antibody, FITC conjugated be integrated with other technologies for comprehensive immune function analysis?

Integrating TAP2 Antibody, FITC conjugated with complementary technologies creates powerful research platforms:

• Single-cell multi-omics integration:

  • Combine flow cytometry using TAP2 Antibody, FITC conjugated with single-cell RNA sequencing to correlate protein expression with transcriptional profiles.

  • Implement CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) using oligonucleotide-tagged antibodies alongside TAP2 detection to simultaneously measure multiple protein markers and transcriptomes.

• Advanced microscopy techniques:

  • Apply super-resolution microscopy (STORM, STED) with TAP2 Antibody, FITC conjugated to visualize nanoscale organization of the peptide loading complex.

  • Utilize live-cell imaging with TAP2 fusion proteins to complement fixed-cell antibody staining, providing dynamic information about TAP2 trafficking.

• Functional assays correlation:

  • Pair TAP2 expression analysis with peptide transport assays using fluorescent peptide substrates to directly correlate expression with functional activity.

  • Integrate with MHC-I peptide elution and mass spectrometry to connect TAP2 expression levels with the presented peptide repertoire.

• High-content screening applications:

  • Develop automated high-content screening workflows using TAP2 Antibody, FITC conjugated to assess the impact of drug candidates or genetic perturbations on antigen presentation pathways.

  • Combine with luminescent reporters of MHC-I surface expression for multiplexed readouts of antigen presentation pathway integrity.

• In vivo imaging extension:

  • Translate in vitro findings to animal models using adoptively transferred cells labeled with TAP2 Antibody, FITC conjugated for short-term tracking.

  • Develop xenograft models with fluorescent TAP2 reporters to monitor antigen presentation machinery in tumor microenvironments.

By integrating these complementary approaches, researchers can build a comprehensive understanding of TAP2 function in health and disease, particularly in contexts like cancer immunotherapy where antigen presentation efficiency is critical for treatment efficacy .

What emerging research areas might benefit from application of TAP2 Antibody, FITC conjugated?

TAP2 Antibody, FITC conjugated can advance several emerging research frontiers:

• Cancer immunotherapy biomarker development:

  • Screen patient tumor samples for TAP2 expression to predict response to immune checkpoint inhibitors.

  • Monitor changes in TAP2 expression during treatment to identify acquired resistance mechanisms.

  • Investigate combination therapies that might restore TAP2 function in tumors with defective antigen presentation.

• Viral immune evasion mechanisms in emerging pathogens:

  • Characterize TAP2 targeting by novel viral proteins, especially from emerging RNA viruses.

  • Screen compound libraries for molecules that protect TAP2 from viral interference.

  • Develop TAP2-resistant vaccine vectors for enhanced immunogenicity.

• Autoimmune disease pathogenesis:

  • Analyze TAP2 polymorphisms and expression patterns in autoimmune-prone individuals.

  • Investigate altered peptide presentation in tissues affected by autoimmune pathology.

  • Develop therapeutic approaches targeting the antigen presentation pathway in specific autoimmune conditions.

• Tissue-specific antigen presentation biology:

  • Map TAP2 expression across human tissue atlas datasets to identify tissue-specific regulation patterns.

  • Correlate with tissue-resident T cell functionality in health and disease.

  • Investigate specialized antigen presentation mechanisms in immune-privileged sites.

• Aging and immunosenescence research:

  • Track age-related changes in TAP2 expression and function across immune cell subsets.

  • Correlate with altered vaccine responses and infection susceptibility in elderly populations.

  • Develop interventions to restore optimal antigen presentation in aging.

• Extracellular vesicle-mediated antigen presentation:

  • Investigate TAP2 incorporation into exosomes and other extracellular vesicles.

  • Study the transfer of antigen presentation machinery between cells via vesicular transport.

  • Develop engineered vesicles with enhanced TAP function for immunotherapy applications.

These emerging areas represent opportunities where TAP2 Antibody, FITC conjugated can provide valuable insights into fundamental immunological processes with translational potential .