TAP2 Antibody, HRP conjugated

What is the primary function of TAP2 in cellular biology?

TAP2 forms a complex with TAP1 to mediate unidirectional translocation of peptide antigens from the cytosol to the endoplasmic reticulum for loading onto MHC class I molecules. This transport mechanism uses the chemical energy of ATP to export peptides against the concentration gradient. During the transport cycle, TAP2 alternates between an "inward-facing" state with the peptide binding site facing the cytosol and an "outward-facing" state with the site facing the ER lumen . This mechanism is critical for immune surveillance, allowing cells to present intracellular peptide antigens to CD8+ T cells.

What experimental applications are validated for TAP2 antibody, HRP conjugated?

Based on current research validation, TAP2 antibody, HRP conjugated has been specifically tested and validated for ELISA applications . For broader experimental applications, non-conjugated TAP2 antibodies have demonstrated utility in Western blotting, immunoprecipitation, flow cytometry (intracellular), immunocytochemistry/immunofluorescence, and immunohistochemistry (paraffin-embedded sections) . The HRP conjugation provides direct enzymatic detection capability, eliminating the need for secondary antibody incubation steps in applicable assays.

How should TAP2 antibody, HRP conjugated be stored to maintain optimal activity?

For optimal preservation of antibody activity, store TAP2 antibody, HRP conjugated at -20°C or -80°C upon receipt. The antibody formulation typically contains preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4) . Avoid repeated freeze-thaw cycles as these can significantly reduce antibody performance by promoting protein denaturation and aggregation. For working stocks, small aliquots at working concentration can be stored at 4°C for short-term use (1-2 weeks).

How can I determine the optimal working concentration of TAP2 antibody, HRP conjugated for my ELISA experiments?

To determine the optimal working concentration, perform a titration experiment using:

• Prepare serial dilutions of the antibody (typical range: 1:100 to 1:10,000)

• Use positive controls (sample with known TAP2 expression, e.g., IFN-γ stimulated cells) and negative controls (TAP2 knockout samples or isotype controls)

• Plot signal-to-noise ratio against antibody concentration

• Select the concentration that gives maximum signal with minimal background

For TAP2 detection specifically, consider that expression is upregulated in response to IFN-gamma treatment , which can be used as a positive control condition. The antibody's protein G purification (>95% purity) provides high specificity , but validation using knockout samples is still recommended for determining background signal thresholds.

What are the critical steps in sample preparation when using TAP2 antibody, HRP conjugated for protein detection?

Optimal sample preparation involves:

• For cell/tissue lysates: Use non-boiling conditions as boiling may cause TAP2 protein aggregates

• Include protease inhibitors in lysis buffers to prevent degradation

• For membrane proteins like TAP2, use detergent-based lysis buffers (e.g., RIPA with 0.1-0.5% SDS)

• Quantify protein concentration and standardize loading amounts

• For TAP2 specifically, samples treated with IFN-gamma (10 ng/ml for 16 hours) show enhanced expression

• For blocking/dilution, 5% non-fat dry milk in TBST has been validated for TAP2 antibody applications

Importantly, TAP2 typically appears at approximately 75 kDa on Western blots , which should be used as a reference point for confirming specific detection.

How can the specificity of TAP2 antibody, HRP conjugated be verified in complex experimental systems?

For rigorous validation of specificity:

• Genetic approaches: Compare staining in wild-type versus TAP2 knockout cells (validated approach showing no staining in TAP2 knockout HeLa cells)

• Competitive blocking: Pre-incubate antibody with recombinant TAP2 protein (specifically amino acids 473-615, which was used as the immunogen)

• Induction experiments: Compare signal between baseline and IFN-gamma treated cells (TAP2 expression is upregulated with IFN-gamma treatment)

• Multi-antibody validation: Compare results with alternative TAP2 antibodies targeting different epitopes

• Mass spectrometry: Confirm identity of immunoprecipitated proteins

This multi-faceted approach ensures that signals detected are specifically from TAP2 rather than from non-specific binding or cross-reactivity.

What are the technical considerations for multiplex immunofluorescence applications involving TAP2?

When designing multiplex experiments:

• Antibody panel selection: Choose antibodies raised in different host species to avoid cross-reactivity

• Subcellular localization reference: Since TAP2 localizes to the endoplasmic reticulum, co-staining with ER markers (e.g., KDEL, as validated) provides spatial reference

• Sequential staining protocol: For multiple rabbit antibodies, consider sequential staining with thorough blocking between steps

• Signal separation: For HRP-conjugated antibodies in multiplex experiments, use tyramide signal amplification with distinct fluorophores

• Controls: Include single-stain controls to verify absence of spectral overlap

In confocal microscopy applications, TAP2 antibody has been validated to show endoplasmic reticulum staining in wildtype cells and no staining in TAP2 knockout cells , providing a clear benchmark for expected subcellular distribution.

What are the most common causes of false negatives when using TAP2 antibody, HRP conjugated, and how can they be resolved?

Common causes of false negatives include:

• Insufficient target expression: TAP2 expression varies by cell type and condition; consider IFN-gamma treatment (10 ng/ml for 16 hours) to upregulate expression

• Improper sample preparation: For membrane proteins like TAP2, ensure adequate membrane solubilization; avoid boiling samples as this may cause protein aggregation

• Antibody degradation: Store according to manufacturer recommendations (-20°C or -80°C); avoid repeated freeze-thaw cycles

• Incorrect buffer conditions: Verify pH and ionic strength of buffers; use recommended dilution buffer (5% NFDM/TBST for western applications)

• HRP inactivation: Avoid sodium azide in buffers when using HRP-conjugated antibodies as it inhibits peroxidase activity

To resolve these issues, implement positive controls (IFN-gamma treated cells, tissues with known TAP2 expression like tonsil tissue) and optimize detection conditions with longer exposure times if necessary.

How does TAP2 antibody performance compare across different tissue and cell types?

Based on validated applications:

For tissues with potential low expression, signal amplification systems or longer incubation times may be necessary. Expression patterns correlate with immune function tissues, with highest expression in lymphoid organs.

How should quantitative data from TAP2 antibody, HRP conjugated experiments be normalized for comparative studies?

For reliable quantitative comparisons:

• Loading controls: Normalize TAP2 signal to housekeeping proteins (β-actin, GAPDH) for western blots

• Reference standards: Include recombinant TAP2 protein standard curve for absolute quantification in ELISA

• Multiple normalization approaches: Compare results using different reference genes/proteins

• Biological variation accounting: For patient samples, consider demographic factors that may influence TAP2 expression

• Technical replicates: Minimum of three technical replicates to account for assay variation

When analyzing IFN-gamma-induced expression changes, calculate fold-change relative to untreated samples rather than absolute values, as baseline expression can vary between experimental systems.

What are the key considerations when interpreting TAP2 detection results in the context of MHC class I antigen presentation research?

Critical interpretation factors include:

• Functional correlation: TAP2 expression alone doesn't guarantee functional peptide transport; consider co-expression with TAP1 and other peptide loading complex components

• Regulatory context: IFN-gamma upregulates TAP2 expression , but other inflammatory mediators may have different effects

• Genetic variation: Human TAP2 polymorphisms can affect peptide selectivity and transport efficiency

• Subcellular localization: Proper TAP2 function requires correct ER localization, which should be verified by co-localization with ER markers (e.g., KDEL)

• Pathological interpretation: Decreased TAP2 expression is associated with immune evasion in some cancers and viral infections

For comprehensive understanding of antigen presentation defects, consider parallel assessment of MHC class I surface expression, which depends on functional TAP2/TAP1-mediated peptide transport.

How can TAP2 antibody, HRP conjugated be utilized in studying viral immune evasion mechanisms?

For investigating viral evasion strategies:

• Comparative expression analysis: Measure TAP2 levels in infected versus uninfected cells using the HRP-conjugated antibody in ELISA or western blot formats

• Kinetic studies: Track TAP2 degradation following viral infection through time-course experiments

• Interaction studies: Combine with co-immunoprecipitation to identify viral proteins targeting the TAP complex

• Functional correlation: Correlate TAP2 expression levels with surface MHC class I expression and CD8+ T cell recognition

• Rescue experiments: Test if overexpression of TAP2 can overcome viral immune evasion

Several viruses (HSV, CMV, adenovirus) produce proteins that interfere with TAP function. Using the antibody to monitor TAP2 levels can provide insights into the molecular mechanisms underlying this interference.

What methodological approaches can be used to study TAP2 in patient-derived samples for immunodeficiency or autoimmune disease research?

For clinical research applications:

• Immunohistochemistry: TAP2 antibody has been validated for IHC-P applications in tissues like kidney , allowing assessment in biopsy samples

• Flow cytometry: Intracellular staining for TAP2 in peripheral blood mononuclear cells can identify deficiencies

• Functional transport assays: Combine TAP2 expression data with functional peptide transport assays in patient cells

• Genetic correlation: Integrate antibody-based detection with sequencing data to correlate expression with genetic variants

• Therapeutic monitoring: Track changes in TAP2 expression during immunomodulatory therapies

For rare TAP2 deficiency conditions, establishing baseline expression in healthy control samples is critical, with age and sex-matched controls recommended due to potential variation in expression patterns.