SERPINA1 Antibody

What is SERPINA1 and why is it important in research?

SERPINA1, also known as alpha-1 antitrypsin (AAT), is a serine protease inhibitor that plays a crucial role in regulating protease activity and inflammation in the body. It's primarily synthesized by hepatocytes, with smaller amounts produced by intestinal epithelial cells, neutrophils, pulmonary alveolar cells, and macrophages . SERPINA1 is highly polymorphic, with more than 100 variants described in scientific databases . The protein targets elastase, plasmin, thrombin, trypsin, chymotrypsin, and plasminogen activator . Dysregulation of SERPINA1 has been implicated in several diseases, including chronic obstructive pulmonary disease (COPD), emphysema, and liver disease, making it an important target for therapeutic interventions and diagnostic assays .

What are the commonly available types of SERPINA1 antibodies?

SERPINA1 antibodies are available in multiple formats with different characteristics to suit various research applications:

The choice between monoclonal and polyclonal depends on your research needs - monoclonals offer higher specificity and reproducibility, while polyclonals provide enhanced signal through multiple epitope binding .

What are the most common applications for SERPINA1 antibodies?

SERPINA1 antibodies have been validated for numerous laboratory techniques:

How should I select the appropriate SERPINA1 antibody for my specific research application?

Selection of the optimal SERPINA1 antibody requires careful consideration of several factors:

• Target species concordance: Ensure the antibody reactivity matches your experimental model. For human samples, antibodies like MAB1268 and AF1268 have demonstrated high specificity , while for cross-species work, options like 16382-1-AP show reactivity with human, mouse, and rat samples .

• Application validation: Verify the antibody has been validated for your specific application. For example, antibody PB10097 is specifically guaranteed for Western blot applications , while others like 16382-1-AP are validated for multiple applications including WB, IHC, and IF .

• Epitope location: Consider epitope location based on your research question. Different antibodies target different regions of SERPINA1:

  • N-terminal region (AA 25-315): CAB12481 and others

  • Middle region (AA 165-444): ABIN7166367

  • C-terminal region: Other specific antibodies

• Validation evidence: Review provided validation data, such as Western blot images showing expected molecular weight (47-65 kDa for SERPINA1) and positive controls in relevant tissues (liver, lung, kidney) .

• Technical requirements: Consider concentration, formulation, and storage requirements based on your laboratory conditions .

What positive and negative controls should I use when working with SERPINA1 antibodies?

Positive Controls:

• Cell lines: HepG2 human hepatocellular carcinoma cells are widely used as positive controls for SERPINA1 expression

• Tissue samples: Human liver, lung, kidney tissues, and human plasma consistently show strong SERPINA1 expression

• Recombinant protein: Purified recombinant SERPINA1 at known concentrations for standard curves in quantitative assays

Negative Controls:

• Knockout models: SERPINA1 knockout HepG2 cell line provides an excellent negative control, as demonstrated in immunocytochemistry validation studies

• Isotype controls: Matching IgG from the same species as the primary antibody but without specific target binding

• Secondary-only controls: Omitting primary antibody to check for non-specific binding of secondary antibodies

How can I validate the specificity of a SERPINA1 antibody?

Multi-level validation ensures antibody specificity:

• Knockout/knockdown verification: The gold standard for specificity is demonstrating absence of signal in SERPINA1 knockout models. For example, Serpin A1 is specifically detected in HepG2 parental cells but not in Serpin A1 knockout HepG2 cells .

• Cross-reactivity testing: Some antibodies have been verified to show no cross-reactivity with related serpins. For instance, certain anti-human SERPINA1 antibodies show no cross-reactivity with recombinant human Serpin A3, A4, or A5 in Western blots .

• Multiple detection methods: Confirm expression using orthogonal techniques (WB, IHC, and IF) to build confidence in antibody specificity .

• Mass spectrometry correlation: For definitive validation, compare antibody-based detection with MS identification of the target protein.

What are the best practices for using SERPINA1 antibodies in immunohistochemistry?

For optimal IHC results with SERPINA1 antibodies:

• Antigen retrieval optimization: Different antibodies require specific retrieval methods:

  • For antibody 16382-1-AP, antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may be used as an alternative

  • For MAB1268, heat-induced epitope retrieval using basic pH retrieval reagents is recommended

• Dilution optimization: Start with the manufacturer's recommended range (typically 1:400-1:1600 for SERPINA1 antibodies) and titrate to determine optimal signal-to-noise ratio

• Detection system selection: For sensitive detection, polymer-based systems like Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody have shown excellent results with SERPINA1 antibodies

• Subcellular localization awareness: SERPINA1 typically shows cytoplasmic localization in positive cells, particularly in hepatocytes and epithelial cells

• Tissue-specific considerations: When examining liver tissue, be aware that SERPINA1 expression can vary based on pathological state, providing important diagnostic information in conditions like alpha-1 antitrypsin deficiency

How should I troubleshoot weak or non-specific signals when using SERPINA1 antibodies in Western blotting?

For weak signals:

• Protein loading: SERPINA1 is abundant in liver and plasma samples but may require higher protein loading from other tissues

• Transfer efficiency: Use optimized transfer conditions for glycoproteins like SERPINA1 (47-65 kDa)

• Antibody concentration: Consider increasing antibody concentration (e.g., from 1:2000 to 1:500)

• Detection system: Switch to more sensitive detection systems like enhanced chemiluminescence

• Buffer composition: Try Immunoblot Buffer Group 1 as recommended for certain SERPINA1 antibodies

For non-specific signals:

• Blocking optimization: Increase blocking time or concentration (typically 5% non-fat milk/TBS for 1.5 hours works well)

• Washing stringency: Increase washing steps with TBS-0.1% Tween (3 times, 5 minutes each)

• Antibody specificity: Consider antibodies validated against knockout controls

• Sample preparation: Ensure complete denaturation of samples when using reducing conditions

• Molecular weight verification: For SERPINA1, confirm bands at the expected molecular weight range (47-65 kDa, with specific observations of 50-60 kDa for some antibodies)

What considerations are important when designing SERPINA1 knockdown or knockout validation experiments?

When validating SERPINA1 antibodies using knockdown or knockout approaches:

• Cell line selection: HepG2 cells are ideal for validation as they naturally express high levels of SERPINA1 and knockout models are available

• Validation methods: Employ multiple detection methods to confirm knockout:

  • Western blot: Complete absence of the 47-65 kDa band in knockout samples

  • Immunofluorescence: Loss of cytoplasmic staining in knockout cells

  • qPCR: Confirmation of transcript depletion

• Controls: Include both wild-type and heterozygous samples when possible to demonstrate dose-dependent detection

• Rescue experiments: Re-expression of SERPINA1 in knockout models should restore antibody detection

• Consideration of SERPINA1 variants: The SERPINA1 gene is highly polymorphic with more than 100 variants , so ensure your knockout strategy accounts for potential variant-specific effects

How can I interpret differences in SERPINA1 molecular weight observed across different experimental systems?

SERPINA1 exhibits molecular weight variations that can be attributed to several factors:

• Glycosylation patterns: SERPINA1 contains three N-glycosylation sites (N70, N107, N271) that can result in heterogeneous migration patterns

• Observed weight ranges: Research has documented SERPINA1 appearing at:

  • 47-51 kDa (theoretical calculated weight)

  • 50-60 kDa in human plasma and tissue lysates

  • 62-65 kDa in certain detection systems like Simple Western

• Experimental factors affecting observed weight:

  • Gel percentage: Lower percentage gels show higher apparent MW

  • Reduction conditions: Fully reduced samples may migrate differently

  • Buffer systems: Different electrophoresis buffers affect migration

  • Detection methods: Traditional Western blot vs. capillary electrophoresis (Simple Western)

• Physiological explanations:

  • SERPINA1 can form dimers or higher-order complexes

  • Post-translational modifications vary by tissue origin

  • Some variants affect protein migration patterns

When comparing results across studies, it's essential to consider these technical variations rather than assuming discrepancies represent experimental errors.

What are the known challenges in differentiating SERPINA1 variants with antibody-based methods?

Detecting specific SERPINA1 variants presents several challenges:

• Epitope conservation: Most commercial antibodies target conserved regions of SERPINA1 and cannot distinguish between common variants like M, S, Z, or rare variants

• Single amino acid mutations: Many pathogenic variants differ by only a single amino acid substitution, making them antigenically similar (e.g., PiSDonosti (S+Ser14Phe), PiTijarafe (Ile50Asn))

• Conformational differences: Some variants affect protein folding rather than epitope sequence, requiring conformation-specific antibodies

• Alternative methods required: For definitive variant identification:

  • Complete gene sequencing is necessary to identify the more than 100 SERPINA1 variants

  • Mass spectrometry can identify variant-specific peptides

  • Isoelectric focusing can separate some variants based on charge differences

• Null variants: Some mutations result in no protein production (Q0 variants), requiring genetic rather than antibody-based detection

How do I reconcile contradictory results when comparing SERPINA1 detection across different antibodies?

When faced with discrepant results between different SERPINA1 antibodies:

• Epitope mapping comparison: Different antibodies target different regions of SERPINA1:

  • N-terminal antibodies may miss C-terminal processing events

  • Antibodies targeting the reactive center loop may show reduced binding after protease interaction

• Validation hierarchy: Prioritize results from antibodies with the most rigorous validation:

  • Knockout-validated antibodies provide highest confidence

  • Multiple application-validated antibodies strengthen confidence

  • Antibodies showing expected tissue distribution patterns

• Technical explanations:

  • Sample preparation differences (denaturing vs. native conditions)

  • Buffer incompatibilities

  • Detection system sensitivities

• Biological explanations:

  • SERPINA1 can exist in multiple conformational states

  • Polymeric forms in certain disease states may mask epitopes

  • Tissue-specific post-translational modifications

• Orthogonal validation: To resolve discrepancies, employ antibody-independent methods like mass spectrometry or functional assays for elastase inhibition.

How can SERPINA1 antibodies be employed in studying the molecular pathogenesis of alpha-1 antitrypsin deficiency?

SERPINA1 antibodies are essential tools for investigating AATD pathogenesis:

• Detection of polymerized forms: Specialized conformation-specific antibodies can distinguish between monomeric and polymerized SERPINA1, crucial for studying Z variant accumulation in hepatocytes

• Cellular trafficking analysis: Using immunofluorescence with markers for ER, Golgi, and secretory vesicles, researchers can track intracellular trafficking defects of mutant SERPINA1 variants

• Degradation pathway investigation: Antibodies help elucidate whether specific variants undergo proteasomal or autophagic degradation

• Functional domain analysis: Antibodies targeting different domains can assess structural integrity and functional capacity of variant proteins:

  • Reactive center loop accessibility

  • Conformational changes during inhibitory function

  • Polymerization-prone regions

• Novel variant characterization: For newly discovered variants (like the seven novel missense variants described in Spanish patients), antibodies help determine:

  • Intracellular polymer formation

  • Secretion efficiency

  • Antielastase activity

What are the emerging methodologies for studying SERPINA1 in complex biological systems?

Cutting-edge approaches utilizing SERPINA1 antibodies include:

• Multiplexed imaging:

  • Mass cytometry imaging (IMC) using metal-conjugated SERPINA1 antibodies for simultaneous detection of multiple markers

  • Multiplexed immunofluorescence to study SERPINA1 in the context of inflammatory mediators

• Live-cell imaging:

  • Antibody fragments conjugated to fluorescent proteins for tracking SERPINA1 trafficking in real-time

  • FRET-based approaches to study SERPINA1 interactions with proteases

• Single-cell analysis:

  • Combined antibody-based protein detection with single-cell RNA-seq to correlate SERPINA1 protein expression with transcriptional landscapes

  • Mass cytometry (CyTOF) with SERPINA1 antibodies for high-dimensional analysis of expression in heterogeneous cell populations

• Computational approaches:

  • Integration of antibody-based detection data with bioinformatic tools to predict effects of SERPINA1 variants

  • Structural modeling combined with epitope mapping to design variant-specific antibodies

• Therapeutic development:

  • Using antibodies to screen for compounds that prevent SERPINA1 polymerization

  • Antibody-based targeting of intracellular SERPINA1 aggregates for degradation

How can advanced computational tools assist in analyzing the effects of SERPINA1 variants detected with antibody-based methods?

Computational approaches enhance antibody-based SERPINA1 research:

• Structural impact prediction: Computational tools can predict how variants affect protein structure and potentially alter antibody epitopes or function:

  • Predict changes in protein stability and folding

  • Model effects on protease inhibition mechanism

  • Simulate polymerization propensity

• Epitope mapping: Advanced algorithms help map conformational epitopes and predict which variants might affect antibody binding:

  • Identification of surface-exposed residues

  • Prediction of antigenic determinants

  • Analysis of potential cross-reactivity with related serpins

• Integration with genomic data:

  • Correlating antibody-detected protein levels with SERPINA1 gene variants

  • Predicting functional consequences of novel variants through machine learning algorithms

  • Analysis of gene-protein-function relationships in rare SERPINA1 variants

• Systems biology approaches:

  • Network analysis incorporating SERPINA1 protein interactions detected by co-immunoprecipitation

  • Pathway modeling to understand consequences of SERPINA1 dysfunction

  • Multi-omics data integration with antibody-based protein quantification

• Clinical translation:

  • Development of variant-specific diagnostic algorithms

  • Personalized medicine approaches based on SERPINA1 variant profiles

  • Computational drug design targeting specific SERPINA1 variant conformations