Leptin PAT7E10AT Antibody

What is the specificity and reactivity profile of the Leptin PAT7E10AT Antibody?

The PAT7E10AT is a mouse monoclonal antibody that specifically targets human leptin, a key hormone in the neuroendocrine axis regulating appetite and metabolism. This antibody recognizes an epitope within amino acids 22-167 of human leptin, as it was raised against recombinant human leptin protein purified from E. coli expression systems. Current validation confirms its reactivity with human samples, making it suitable for studies investigating human leptin biology . When designing experiments, researchers should consider that while this antibody has high specificity for human leptin, cross-reactivity testing with other species should be performed if working with non-human models.

What applications has the Leptin PAT7E10AT Antibody been validated for?

The PAT7E10AT antibody has been validated for multiple applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

• Immunocytochemistry/Immunofluorescence (ICC/IF)

For each application, specific optimization protocols may be necessary. In ELISA applications, this antibody can be used for developing custom assays to quantify leptin levels or detect leptin-reactive antibodies in biological samples, similar to methods described in studies measuring anti-leptin antibodies .

What are the optimal storage and handling conditions for maintaining antibody activity?

To maintain optimal activity of the PAT7E10AT antibody, researchers should adhere to these storage guidelines:

• Shipping condition: 4°C

• Short-term storage (up to 1 month): 2-8°C

• Long-term storage: -20°C

• Avoid repeated freeze/thaw cycles as they can compromise antibody performance

The antibody is supplied at a concentration of 1 mg/ml in Phosphate Buffered Saline (pH 7.4) containing 0.02% Sodium Azide and 10% Glycerol. This formulation helps maintain stability during storage. When planning long-term studies, it is advisable to aliquot the antibody into single-use volumes to minimize freeze/thaw cycles.

What controls should be included when using the PAT7E10AT Antibody in experiments?

For rigorous experimental design, include the following controls:

• Isotype controls: Mouse IgG2b [PLRV219] (A86740) or Mouse IgG2b [MPC-11] (A86389) at matching concentrations to rule out non-specific binding

• Negative controls:

  • Primary antibody omission

  • Samples known to be negative for leptin expression

  • Blocking peptide competition (using recombinant human leptin)

• Positive controls:

  • Human adipose tissue extracts

  • Recombinant human leptin protein

  • Cell lines known to express leptin (e.g., differentiated adipocytes)

What dilution ranges are recommended for different applications of PAT7E10AT Antibody?

Based on similar leptin antibody protocols and standard monoclonal antibody usage, the following dilution ranges are recommended:

When establishing optimal conditions for your specific experimental system, perform a dilution series to determine the concentration that provides the best signal-to-noise ratio. For ELISA applications, researchers have reported using similar antibodies at 1:1000 dilution with 2-hour incubation periods .

How should samples be prepared for optimal detection of leptin using PAT7E10AT Antibody?

Sample preparation significantly impacts experimental success:

For Western Blot:

• Protein extraction should use buffers containing protease inhibitors

• For serum/plasma samples, consider pre-clearing with Protein A/G to reduce background

• Use optimized protein loading (15-30 μg for tissue lysates)

• Consider sample denaturation conditions carefully (reducing vs. non-reducing)

For ICC/IF:

• Fixation with 4% paraformaldehyde (10-15 minutes) maintains epitope accessibility

• Permeabilization with 0.1-0.3% Triton X-100 (10 minutes)

• Blocking with 5% normal serum (from the species of secondary antibody)

• Overnight primary antibody incubation at 4°C may yield better results than shorter incubations

For ELISA:

• Coating plates with recombinant human leptin (1 mg/ml) for 24 hours at 4°C produces reliable results

• Using appropriate dilution buffers at pH 7.4 (normal conditions) or pH 8.9 (dissociative conditions) when measuring different fractions of leptin-reactive antibodies

What secondary antibodies are most compatible with PAT7E10AT Antibody?

Since PAT7E10AT is a mouse IgG2b isotype antibody with kappa light chains, the following secondary antibodies are recommended:

• Goat Anti-Mouse IgG H&L Antibody (AP) (A301438)

• Goat Anti-Mouse IgG H&L Antibody (Biotin) (A301439)

• Goat Anti-Mouse IgG H&L Antibody (FITC) (A301669)

• Goat Anti-Mouse IgG H&L Antibody (HRP) (A301445)

For ELISA applications, HRP-conjugated secondary antibodies are commonly used at 1:8000 dilution, as described in similar protocols for detecting anti-leptin antibodies . For visualization in Western blots or immunofluorescence studies, select a secondary antibody conjugated to a detection system appropriate for your instrumentation.

What are common causes of weak or no signal when using PAT7E10AT Antibody?

Several factors can contribute to suboptimal signal:

• Epitope masking or destruction:

  • Over-fixation can cross-link proteins excessively

  • Inappropriate antigen retrieval methods

  • Suboptimal sample preparation (e.g., over-heating during SDS-PAGE)

• Technical issues:

  • Inactive antibody due to improper storage or excessive freeze/thaw cycles

  • Insufficient antibody concentration or incubation time

  • Inappropriate blocking agents that may cross-react with the antibody

  • Buffer incompatibility affecting antibody binding

• Biological factors:

  • Low abundance of target protein

  • Post-translational modifications affecting epitope recognition

  • Expression level variations based on physiological state (e.g., leptin expression can vary with nutritional status)

Optimization strategies:

• Titrate antibody concentration

• Extend incubation time (e.g., overnight at 4°C)

• Test different antigen retrieval methods

• Use signal amplification systems (e.g., biotin-streptavidin)

• For Western blots, try both reducing and non-reducing conditions

How can background be reduced when using PAT7E10AT in immunoassays?

Excessive background is a common challenge that can be addressed through these approaches:

For Western Blot:

• Increase blocking time (1-2 hours) with 5% non-fat dry milk or BSA

• Add 0.1-0.3% Tween-20 to washing and antibody dilution buffers

• Increase number and duration of washes (5-6 washes, 10 minutes each)

• Pre-adsorb primary antibody with proteins from the species being tested

• Use more stringent blocking agents like fish gelatin or commercially available blocking reagents

For ICC/IF:

• Include 0.1-0.3% Triton X-100 in blocking solution

• Add 10% normal serum from secondary antibody species to blocking buffer

• Use 0.05% Tween-20 in wash buffers

• Include appropriate isotype controls

• Consider autofluorescence quenching treatments

For ELISA:

• Optimize plate coating conditions

• Ensure adequate blocking (2% BSA in PBS)

• Use 0.05% Tween-20 in wash buffers

• Optimize sample dilutions to stay within the linear range

• Set appropriate blank OD values (below 0.1) to confirm absence of unspecific binding

How can researchers validate the specificity of PAT7E10AT in their experimental system?

Validation should include multiple approaches:

• Peptide competition assay:

  • Pre-incubate the antibody with excess recombinant human leptin

  • A specific antibody will show reduced or eliminated signal

• Knockdown/Knockout controls:

  • Use leptin siRNA or CRISPR/Cas9 systems to create negative controls

  • Compare signal in wild-type vs. leptin-deficient samples

• Correlation with other detection methods:

  • Compare results with other validated anti-leptin antibodies

  • Correlate protein detection with mRNA levels (qPCR)

• Western blot analysis:

  • Confirm detection of a band at the expected molecular weight (~16 kDa for leptin)

  • Absence of non-specific bands

• Positive and negative tissue controls:

  • Human adipose tissue (positive)

  • Tissues known not to express leptin (negative)

How can PAT7E10AT be used to study leptin-reactive antibodies in metabolic disorders?

The PAT7E10AT antibody can be instrumental in developing assays to study leptin-reactive antibodies in various patient populations:

• Development of custom ELISA systems:

  • Coat plates with recombinant human leptin (1 mg/ml for 24h at 4°C)

  • Use different buffer conditions to detect:

    • Free leptin-reactive antibodies (normal pH 7.4 buffer)

    • Total leptin-reactive antibodies (dissociative pH 8.9 buffer)

    • Calculate immune complexes percentage: 100 – (Free OD/Total OD) × 100

• Analytical considerations:

  • Standardize serum dilutions (1:1000 has been effective in previous studies)

  • Optimize incubation times (2h is commonly used)

  • Select appropriate detection antibody dilutions (1:8000 for anti-human IgG)

• Research applications:

  • Compare leptin-reactive antibody levels across different BMI categories

  • Correlate antibody levels with metabolic risk indexes (HOMA-IR, HOMA-β, AIP)

  • Investigate age-related differences in antibody production (children vs. adolescents vs. adults)

  • Study potential protective roles of leptin-reactive antibodies in insulin resistance

Research has shown that IgG anti-leptin antibodies correlate with BMI differently in children and adolescents, suggesting age-dependent effects on antibody production in response to obesity .

What protocols exist for using PAT7E10AT in studying leptin's role in autoimmune conditions?

Recent research has implicated leptin in autoimmune processes, including Systemic Lupus Erythematosus (SLE). The PAT7E10AT antibody can be utilized to investigate these connections:

• Flow cytometry protocols:

  • Use PAT7E10AT to detect intracellular leptin or as a capture antibody

  • Combine with anti-leptin receptor antibodies to investigate signaling

  • Fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor® 647) can be used for detection

• Investigation of leptin signaling in immune cells:

  • Isolate CD4+ T cells from patients and healthy controls

  • Analyze leptin and leptin receptor expression in different T cell subsets

  • Stimulate cells with recombinant leptin and measure downstream signaling

  • Evaluate effects on T cell differentiation and cytokine production

• Translational research applications:

  • Use in humanized mouse models to study leptin's role in autoimmunity

  • Investigate leptin's effects on AMPK activation in immune cells

  • Combine with phospho-flow cytometry to assess leptin-induced signaling pathways

How can PAT7E10AT be integrated with other research tools for comprehensive leptin studies?

Multimodal approaches enhance research depth:

• Multi-antibody strategies:

  • Combine PAT7E10AT with antibodies against leptin receptor

  • Use with phospho-specific antibodies (e.g., phospho-AMPK) to track signaling

  • Pair with antibodies against downstream effectors (STAT3, JAK2, etc.)

• Hybrid techniques:

  • Immunoprecipitation with PAT7E10AT followed by mass spectrometry

  • ChIP-Seq to identify leptin-regulated genes

  • Proximity ligation assays to study leptin-receptor interactions

• Advanced imaging:

  • Multiplex immunofluorescence with PAT7E10AT and other markers

  • Super-resolution microscopy to study leptin distribution

  • Live-cell imaging with labeled PAT7E10AT fragments

• Systems biology approaches:

  • Integration with transcriptomics and proteomics data

  • Network analysis of leptin signaling pathways

  • Computational modeling of leptin-mediated effects

What statistical approaches are recommended for analyzing data from PAT7E10AT-based assays?

Appropriate statistical analysis enhances data interpretation:

• For ELISA and other quantitative assays:

  • Verify data distribution using D'Agostino–Pearson normality test

  • Use parametric tests (ANOVA, t-test) for normally distributed data

  • Apply non-parametric alternatives (Kruskal-Wallis, Mann-Whitney) for non-normal distributions

  • Perform appropriate post-hoc tests (Tukey's or Dunn's) for multiple comparisons

• For correlation analyses:

  • Use Pearson's correlation for normally distributed data

  • Apply Spearman's correlation for non-parametric data

  • Consider multivariate linear regression to analyze associations between multiple variables

• Quality control measures:

  • Use duplicate or triplicate samples (aim for <15% variation)

  • Include appropriate blanks and subtract blank OD values

  • Establish standard curves with recombinant leptin for quantification

  • Set acceptance criteria for assay validity

How should researchers interpret differences in leptin-reactive antibody profiles between subject groups?

Interpretation requires careful consideration of multiple factors:

• Biological significance:

  • Age-dependent effects (children vs. adolescents)

  • BMI-dependent patterns (normal weight vs. overweight vs. obesity)

  • Correlation with metabolic parameters (HOMA-IR, HOMA-β, AIP)

  • Potential protective or pathogenic roles

• Technical considerations:

  • Distinguish between free antibodies, total antibodies, and immune complexes

  • Consider antibody affinity and avidity effects

  • Evaluate the impact of sample handling and storage

Research has shown that IgG anti-leptin antibody levels correlate positively with BMI in adolescents but negatively in children, suggesting complex age-dependent immunological responses to metabolic states .

The relationship between leptin-reactive antibodies and insulin resistance (negative correlation with HOMA-IR in both children and adolescents) suggests potential protective roles against metabolic dysfunction .

What reference ranges should researchers consider when interpreting PAT7E10AT-based assay results?

While standardized reference ranges for leptin-reactive antibodies are still evolving, researchers should consider:

• Population-specific baselines:

  • Age groups (children, adolescents, adults)

  • BMI categories (normal weight, overweight, obesity)

  • Sex differences (male vs. female)

  • Health status (healthy vs. metabolic disorders)

• Internal normalization approaches:

  • Express results as fold-change relative to control groups

  • Use percentile ranks within study population

  • Consider immune complex percentages rather than absolute values

  • Normalize to total IgG levels to account for individual variations

• Technical benchmarks:

  • Establish assay-specific cutoffs based on control populations

  • Use receiver operating characteristic (ROC) curves to determine optimal thresholds

  • Consider Z-scores for standardization across different studies