TJP2 Antibody, HRP conjugated

What is the optimal application for TJP2 antibody, HRP conjugated?

TJP2 antibody, HRP conjugated is primarily optimized for ELISA applications. The direct HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows. While the HRP-conjugated version is specifically validated for ELISA, other variants of TJP2 antibodies without HRP conjugation can be used for Western blotting, immunohistochemistry (IHC), immunofluorescence (IF), and immunocytochemistry (ICC) .

What are the recommended positive control samples for validating TJP2 antibody performance?

For positive controls, human cell lines such as A431, HepG2, MCF-7, U2OS, and HeLa have demonstrated consistent TJP2 expression in Western blot applications. For tissue controls, human liver and intestinal tissue, as well as mouse and rat liver and kidney tissues have shown reliable TJP2 immunoreactivity . When optimizing new experimental conditions, including at least one validated positive control is essential for confirming antibody functionality.

How should I prepare samples for optimal TJP2 detection in ELISA?

For optimal detection in ELISA applications:

• For tissue homogenates: Process fresh tissue samples on ice, homogenize in PBS (pH 7.4) with protease inhibitors, centrifuge at 10,000-15,000g for 20-30 minutes at 4°C, and collect the supernatant

• For cell lysates: Wash cells with ice-cold PBS, lyse using buffer containing 1% Triton X-100, 150mM NaCl, 50mM Tris-HCl (pH 8.0) and protease inhibitors, incubate on ice for 30 minutes with occasional vortexing, centrifuge at 12,000g for 15 minutes at 4°C, then collect the supernatant

Sample concentration should fall within the detection range of 0.16-10 ng/mL for optimal results with commercial ELISA kits .

What is the binding specificity of commonly available TJP2 antibodies, and how does epitope location affect experimental outcomes?

Different TJP2 antibodies target distinct epitopes within the protein. For example:

• AA 1165-1183 (C-terminal region): Targets the C-terminal domain, ideal for detecting full-length TJP2

• AA 307-669: Targets the central region containing PDZ domains, useful for studying protein-protein interactions

• AA 121-218: Targets the N-terminal region, which can detect alternative splicing variants

The epitope choice affects experimental outcomes significantly. C-terminal antibodies may fail to detect truncated variants, while N-terminal antibodies might detect all isoforms but miss post-translational modifications. To comprehensively study TJP2, researchers should consider using antibodies targeting different epitopes based on their specific research questions .

What are the recommended storage and handling conditions to maintain antibody activity?

For optimal antibody performance:

• Store lyophilized antibody at -20°C upon receipt

• After reconstitution, store at 4°C for up to one month for regular use

• For long-term storage, aliquot and store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles (no more than 3 cycles)

• For HRP-conjugated antibodies, ensure storage in glycerol-containing buffer (typically 50% glycerol, 0.01M PBS, pH 7.4) with preservative (such as 0.03% Proclin 300)

Working dilutions should be prepared fresh on the day of experiment to maintain optimal signal-to-noise ratio .

How can researchers validate specificity when working with TJP2 antibody in a new experimental system?

To validate antibody specificity in a new system:

• Perform Western blot to confirm a single band at the expected molecular weight (~150 kDa, though the calculated molecular weight is ~131 kDa)

• Include a peptide competition assay using the immunizing peptide sequence

• Test in known positive and negative control samples

• Consider siRNA knockdown or CRISPR knockout of TJP2 as definitive negative controls

• For HRP-conjugated antibodies, include controls without primary antibody to assess potential non-specific binding

When working with disease models where TJP2 might be affected, such as cholestatic liver disease, validation becomes particularly critical as protein expression patterns may change .

How can I optimize TJP2 detection in challenging tissue samples like liver or intestine where tight junctions are specialized structures?

For optimizing TJP2 detection in challenging epithelial tissues:

• Antigen retrieval is critical - use EDTA buffer (pH 8.0) for heat-mediated retrieval rather than citrate buffer

• For liver tissue, extend the primary antibody incubation to overnight at 4°C to improve sensitivity

• Block with 10% normal goat serum to reduce background staining

• For intestinal samples, a mild permeabilization step (0.1% Triton X-100 for 10 minutes) improves antibody access to tight junctions

• Consider tyramide signal amplification for enhanced sensitivity in tissues with low TJP2 expression

These tissues often require more stringent blocking and washing steps due to endogenous biotin and peroxidase activity .

What methods can differentiate between TJP2 subcellular localization changes versus expression level changes?

To distinguish localization from expression changes:

• Combine subcellular fractionation with Western blotting to quantify TJP2 in membrane, cytoplasmic, and nuclear fractions

• Employ dual-labeling immunofluorescence with markers for tight junctions (e.g., claudins), adherens junctions (e.g., β-catenin), and nuclear markers

• Use confocal microscopy with Z-stack imaging to precisely localize TJP2 in relation to plasma membrane

• Normalize Western blot data to both loading controls and membrane fraction markers

• For quantification, measure intensity at junction sites versus cytoplasmic regions using image analysis software

This approach is particularly important as TJP2 translocation between membrane and nucleus has been implicated in signaling pathways and disease states .

How do mutations in TJP2 affect antibody detection, and what methodological approaches can address this challenge?

TJP2 mutations, particularly protein-truncating mutations, can significantly impact antibody detection:

• For C-terminal antibodies (like those targeting AA 1165-1183), truncating mutations can completely abolish detection as demonstrated in patients with progressive cholestatic liver disease

• Using antibodies targeting different epitopes is crucial when studying potential disease-causing mutations

• Combine mRNA quantification (qPCR) with protein detection to identify nonsense-mediated decay

• When analyzing patient samples with suspected TJP2 mutations, include multiple antibodies targeting different domains (N-terminal, central, and C-terminal)

Research has shown that in patients with TJP2 mutations, immunohistochemical studies with C-terminal antibodies failed to detect the protein, confirming the truncating nature of these mutations .

What is the expected molecular weight of TJP2 in Western blot, and how can researchers address discrepancies between calculated and observed weights?

The calculated molecular weight of TJP2 is approximately 131-134 kDa, but it consistently appears at approximately 150 kDa in SDS-PAGE experiments . This discrepancy is attributed to:

• Post-translational modifications, particularly phosphorylation

• The high proline content affecting protein migration

• Isoform variations due to alternative splicing

To address these discrepancies:

• Always include a positive control sample with known TJP2 expression

• Consider using gradient gels (5-20% SDS-PAGE) for better resolution

• When analyzing phosphorylated forms, include phosphatase-treated samples as controls

• For isoform analysis, reference specific migration patterns in literature

How can TJP2 antibodies be used to investigate tight junction integrity in disease models?

TJP2 antibodies can be valuable tools for investigating tight junction integrity:

• In liver disease models: Combine TJP2 with claudin-1 staining to assess localization defects, as demonstrated in patients with TJP2 mutations where claudin-1 fails to localize properly despite normal protein levels

• In hearing loss models: Monitor Tjp2 expression changes during development, which shows critical temporal patterns (decreasing rapidly between E16.5 and 1 week postnatally)

• In epithelial barrier studies: Use TJP2 antibodies in conjunction with transepithelial electrical resistance (TEER) measurements and permeability assays

• For signaling pathway investigations: Combine with GSK-3β and phospho-GSK-3β (Ser9) detection, as TJP2 overexpression decreases GSK-3β phosphorylation

These approaches can reveal whether junction disruption occurs due to protein mislocalization, degradation, or expression changes .

What controls should be included when using TJP2 antibody, HRP conjugated in multiplexed detection systems?

For multiplexed detection systems:

• Single-stain controls: Include samples stained with each primary antibody alone to assess specificity

• Isotype controls: Use rabbit IgG at the same concentration as the TJP2 antibody to evaluate non-specific binding

• Absorption controls: Pre-incubate antibody with immunizing peptide to confirm specificity

• Cross-reactivity controls: Test for potential cross-reactivity with related tight junction proteins (TJP1/ZO-1, TJP3/ZO-3)

• For HRP-conjugated antibodies specifically, include substrate-only controls to assess potential endogenous peroxidase activity

When performing multiplexed detection with fluorescence, sequential staining rather than simultaneous application may reduce potential cross-reactivity issues .

What are potential causes and solutions for high background when using TJP2 antibody, HRP conjugated in ELISA?

High background in ELISA applications may result from:

Dilution series validation should show OD values following the pattern demonstrated in the standard curve (10.00 ng/mL: 2.096, 5.00 ng/mL: 1.720, 2.50 ng/mL: 1.227, etc.) .

How can researchers interpret inconsistent results between different applications using the same TJP2 antibody?

Inconsistencies between applications may reflect:

• Epitope accessibility differences: Denaturation in Western blot versus native conformation in IF/IHC

• Post-translational modifications affecting epitope recognition differently in various contexts

• Sample preparation variations altering protein presentation

• Antibody concentration optimization differences between techniques

To address these inconsistencies:

• Validate each application independently with appropriate positive controls

• Optimize fixation/permeabilization conditions for each sample type

• Consider epitope retrieval requirements specific to each technique

• For quantitative comparisons across techniques, establish calibration curves using recombinant proteins

When studying TJP2 in different cellular contexts, remember that tight junction composition varies by tissue type, potentially affecting antibody performance .