PAI3 Antibody

What is PAI3 and why is it relevant to research?

PAI3 (Plasminogen Activator Inhibitor 3), also known as Serpin A5 or Protein C inhibitor (PCI), belongs to the serine protease inhibitor superfamily. It functions as a regulatory protein in various biological processes including blood coagulation, tissue remodeling, and reproductive mechanisms. PAI3 has gained research interest due to its roles in protease inhibition pathways and potential implications in disease states. The protein is encoded by the SERPINA5 gene and exhibits tissue-specific expression patterns, making it an interesting target for diverse research applications .

What are the common synonyms and alternative nomenclature for PAI3?

When searching literature or antibody resources, researchers should be aware of multiple designations for this protein:

• Plasminogen Activator Inhibitor 3 (PAI3)

• Serpin A5 (SERPINA5)

• Protein C Inhibitor (PCI or PROCI)

• Plasma Serine Protease Inhibitor

• Acrosomal Serine Protease Inhibitor

• PLANH3

Understanding these alternative names is crucial when conducting literature searches or selecting appropriate antibodies for research applications.

What applications are PAI3 antibodies commonly used for?

PAI3 antibodies are primarily utilized in:

• Western Blot (WB) analysis for protein expression quantification

• Immunocytochemistry (ICC) for cellular localization studies

• ELISA (Enzyme-Linked Immunosorbent Assay) for quantitative detection in biological fluids

• Immunofluorescence for visualization of expression patterns

When selecting a PAI3 antibody, researchers should ensure it has been validated for their specific application. For instance, the rabbit polyclonal PAI3 antibody (A907) has been validated for WB at 1:200 dilution and ICC at 1:50-1:200 dilution ranges .

How should I select between polyclonal and monoclonal PAI3 antibodies?

Selection depends on your experimental goals:

Polyclonal PAI3 antibodies (like A907) recognize multiple epitopes on the PAI3 protein, offering advantages such as:

• Higher sensitivity for detecting low abundance targets

• Greater tolerance to minor protein denaturation or modifications

• Broader reactivity across species (if raised against conserved regions)

These characteristics make polyclonal antibodies suitable for initial characterization studies or when maximum detection sensitivity is required .

Monoclonal PAI3 antibodies provide:

• Higher specificity for a single epitope

• Reduced batch-to-batch variation

• Superior reproducibility in longitudinal studies

• Better suitability for quantitative analyses

Consider using polyclonal antibodies for discovery-phase research and monoclonal antibodies for standardized, quantitative applications requiring consistent performance over time .

What controls should be included in experiments using PAI3 antibodies?

Proper experimental controls are essential for reliable PAI3 antibody research:

• Positive controls: Cell lines or tissues known to express PAI3 (check literature for established expression profiles)

• Negative controls:

  • Isotype control antibodies matching the host species and immunoglobulin class of your PAI3 antibody (e.g., Rabbit IgG controls A82272 or A17360 for rabbit PAI3 antibodies)

  • Cell lines with confirmed absence of PAI3 expression

  • Knockdown/knockout samples when available

• Peptide competition: Pre-incubation of the antibody with immunizing peptide should abolish specific staining

• Secondary antibody only control: To assess non-specific binding of secondary detection reagents

Inclusion of these controls helps distinguish genuine PAI3 signal from technical artifacts and ensures experimental validity.

What are the optimal sample preparation protocols for PAI3 detection?

Sample preparation significantly impacts PAI3 antibody performance:

For Western Blot:

• Use fresh samples whenever possible

• Include protease inhibitors during cell/tissue lysis

• Standard RIPA or TBS-based buffers (as used for A907 antibody) with 0.5% BSA and appropriate detergents are generally suitable

• For membrane-associated protein fraction analysis, consider specialized extraction buffers

• Prevent protein degradation by maintaining cold temperatures throughout processing

For ICC/Immunofluorescence:

• Fixation method affects epitope accessibility; paraformaldehyde (4%) is commonly used

• Compare different permeabilization methods (0.1-0.5% Triton X-100 or saponin) to optimize signal

• Blocking with 5-10% normal serum matching secondary antibody host reduces background

• For PAI3 antibody A907, dilutions between 1:50-1:200 are recommended for ICC applications

How can I validate the specificity of my PAI3 antibody?

Rigorous validation ensures reliable research outcomes:

• Western blot profile analysis: Confirm that the observed band matches the predicted molecular weight of PAI3 (~45-50 kDa)

• Recombinant protein control: Compare reactivity against purified recombinant PAI3

• Genetic approaches:

  • siRNA/shRNA knockdown should reduce antibody signal

  • CRISPR/Cas9 knockout cells provide definitive negative controls

  • Overexpression systems can confirm specificity and sensitivity

• Mass spectrometry validation: Immunoprecipitation followed by mass spectrometry can verify the identity of the pulled-down protein

• Cross-reactivity assessment: Test against related serpins to evaluate potential cross-reactivity, especially important when studying complex biological samples

What approaches can address non-specific binding with PAI3 antibodies?

Non-specific binding can obscure genuine results. Consider these optimization strategies:

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

• Antibody dilution adjustment:

  • Perform titration experiments to identify optimal concentration

  • For PAI3 antibody A907, start with manufacturer-recommended dilutions (1:200 for WB, 1:50-1:200 for ICC)

• Washing modifications:

  • Increase washing duration and frequency

  • Add low concentrations of detergent (0.05-0.1% Tween-20) to wash buffers

• Buffer composition adjustments:

  • Add carrier proteins (0.1-0.5% BSA) to antibody dilution buffer

  • Consider adding 5-10% normal serum from secondary antibody host species

• Antibody pre-adsorption:

  • Pre-incubate with tissues/cells lacking target protein to remove cross-reactive antibodies

How can ELISA be optimized for PAI3 detection in biological fluids?

ELISA represents a valuable quantitative approach for PAI3 research:

• Sample preparation considerations:

  • For serum/plasma samples, dilution ranges typically between 1:10-1:100

  • Sample collection tubes (EDTA, heparin) should be consistent across experiments

  • Centrifugation parameters should effectively remove cellular components

• Detection range optimization:

  • Commercial PAI3 ELISA kits typically provide detection ranges (e.g., 156-10,000 pg/ml)

  • Create standard curves with recombinant PAI3 protein spanning the expected concentration range

  • Include high and low concentration quality controls in each assay

• Cross-reactivity management:

  • Select PAI3-specific ELISA systems with documented absence of cross-reactivity with related proteins

  • Consider pre-clearing samples with protein A/G if interfering factors are suspected

• Data analysis approaches:

  • Use four-parameter logistic regression for standard curve fitting

  • Assess intra-assay and inter-assay CVs (<10% and <15%, respectively)

  • Include positive and negative control samples in each experiment

How do PAI3 antibodies compare with antibodies targeting related serpins?

Understanding differences between serpin antibodies helps experimental design:

PAI3 antibodies should be distinguished from other serpin-targeted reagents like PR3 (Proteinase 3) antibodies, which target a different molecule involved in ANCA-associated vasculitis diagnostics . While both fall within protease-related research, they address different biological pathways.

When designing multi-protein studies involving serpins:

• Confirm each antibody's specificity independently

• Consider using antibodies raised in different host species to enable co-localization studies

• Verify potential cross-reactivity, especially for closely related family members

• Establish separate optimal conditions for each antibody rather than assuming unified protocols

What troubleshooting approaches address common PAI3 antibody experimental issues?

How should researchers interpret contradictory results between different PAI3 antibody clones?

When different PAI3 antibodies yield contradictory results:

• Compare epitope targets:

  • Antibodies recognizing different domains may give disparate results if protein undergoes cleavage or conformational changes

  • Map recognized epitopes through peptide array analysis if discrepancies persist

• Evaluate validation status:

  • Consider the extent of validation for each antibody

  • Prioritize antibodies with documented specificity in relevant applications and tissues

• Reconciliation approaches:

  • Use orthogonal techniques (qPCR, mass spectrometry) to validate expression

  • Employ multiple antibodies targeting different epitopes and compare results

  • Consider knockout/knockdown validation studies

• Result interpretation:

  • Document all antibody information (catalog number, lot, clone) in publications

  • Report contradictory findings transparently rather than selecting only "agreeable" results

  • Consider biologically relevant conditions that might explain different antibody reactivity patterns

How can PAI3 antibodies be employed in studies of protein-protein interactions?

PAI3 functions through interactions with various proteins. To study these interactions:

• Co-immunoprecipitation (Co-IP):

  • Use PAI3 antibodies to pull down the protein complex

  • Verify interaction partners by Western blot or mass spectrometry

  • Compare results in different cellular contexts or disease states

• Proximity ligation assay (PLA):

  • Detect in situ protein interactions at single-molecule resolution

  • Requires PAI3 antibody and antibody against putative interaction partner from different host species

  • Provides spatial information about interaction events

• FRET/BRET approaches:

  • When combined with fluorescently-tagged proteins

  • Can demonstrate direct physical interactions

  • Provides information about interaction dynamics

• Considerations for successful interaction studies:

  • Validate PAI3 antibody epitope accessibility in protein complexes

  • Use gentle lysis conditions to preserve native interactions

  • Include appropriate controls (isotype antibodies, known non-interactors)

What strategies can enhance PAI3 detection sensitivity in low-expression contexts?

When PAI3 expression is limited:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) can enhance detection 10-50 fold

  • Biotin-streptavidin systems provide multivalent signal enhancement

  • Enhanced chemiluminescence substrates for Western blot

• Sample enrichment approaches:

  • Immunoprecipitation to concentrate PAI3 before analysis

  • Subcellular fractionation to isolate compartments with higher PAI3 concentration

  • Optimized extraction buffers to maximize protein recovery

• Detection system selection:

  • For Western blots, select high-sensitivity substrates (femtogram range)

  • For immunofluorescence, consider signal enhancers or specialized detection systems

  • For ELISA, ultrasensitive platforms with lower detection limits (<10pg/ml)

• Optimized imaging settings:

  • Longer exposure times (balanced against background increase)

  • Specialized cameras with higher sensitivity

  • Image stacking and computational enhancement

How can PAI3 antibodies contribute to studies of disease mechanisms?

PAI3's roles in protease regulation make it relevant to various pathologies:

• Methodological approaches:

  • Comparative expression analysis between normal and disease tissues

  • Correlation of PAI3 levels with disease progression markers

  • Evaluation of post-translational modifications in disease states

• Protocol considerations:

  • Standardize tissue collection and processing

  • Include matched controls (adjacent normal tissue, age-matched samples)

  • Consider multiple antibodies targeting different epitopes for verification

• Integration with other methodologies:

  • Combine antibody-based detection with functional assays

  • Correlate protein expression with genetic analysis

  • Interpret results in context of known disease-associated pathways

What are the key considerations when using PAI3 antibodies in biomarker research?

When evaluating PAI3 as a potential biomarker:

• Antibody selection criteria:

  • High specificity confirmed through knockout/knockdown validation

  • Consistent lot-to-lot performance

  • Validated in the specific sample types relevant to the disease

• Standardization approaches:

  • Develop standard operating procedures for sample collection and processing

  • Include calibration standards across experiments

  • Implement quality control measures at each analytical step

• Validation requirements:

  • Technical validation (precision, accuracy, reproducibility)

  • Biological validation (correlation with disease features)

  • Independent cohort validation before clinical implementation

• Confounding factor assessment:

  • Evaluate effects of patient demographics, medications, and comorbidities

  • Determine specificity across related disease conditions

  • Account for biological variability in reference ranges