PYCARD Antibody, HRP conjugated

What is PYCARD protein and why is it significant in immunological research?

PYCARD is a 21.6 kD pro-apoptotic protein containing an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain (CARD) . Its significance stems from its central role as an adaptor molecule in inflammasome formation, where it bridges pattern recognition receptors (PRRs) with procaspase-1, facilitating inflammasome assembly and subsequent caspase-1 activation. This protein promotes caspase-mediated apoptosis, predominantly through activation of caspase-9 . PYCARD may also be a component of the inflammasome complex that includes NALP2, CARD8, and CASP1, contributing to the activation of proinflammatory caspases . In cellular contexts, PYCARD typically resides in the cytoplasm but undergoes redistribution to form distinct aggregates prior to caspase activation, appearing as hollow, perinuclear spherical structures .

What applications are most suitable for HRP-conjugated PYCARD antibodies?

HRP-conjugated PYCARD antibodies are particularly valuable for:

• Western blotting (WB): The enzymatic activity of HRP provides sensitive detection with various substrates (chemiluminescent, colorimetric)

• Immunohistochemistry (IHC): For tissue section analysis with amplified signal detection

• ELISA: Enabling quantitative analysis of PYCARD in complex biological samples

When selecting applications, researchers should consider that validated applications vary by antibody source. For instance, many PYCARD antibodies have been validated for WB (0.5-2.0 μg/ml range), IHC-p, and immunofluorescence applications .

How should PYCARD antibodies be stored and handled to maintain optimal activity?

For maximum stability and performance of PYCARD antibodies:

• Store undiluted antibody solutions between 2°C and 8°C for short-term usage

• For long-term storage, maintain at -20°C with glycerol as a cryoprotectant

• Avoid repeated freeze/thaw cycles which significantly reduce antibody activity

• Most preparations are supplied in phosphate-buffered solutions (pH 7.2-7.4) containing stabilizers like 0.09% sodium azide or protein protectants with 50% glycerol

The typical concentration of purified antibodies ranges from 0.5-1 mg/ml, requiring proper dilution for specific applications . Always centrifuge briefly before opening vials to collect solution at the bottom.

What protocol modifications are necessary when using HRP-conjugated PYCARD antibodies in Western blotting?

When optimizing Western blotting with HRP-conjugated PYCARD antibodies:

• Blocking optimization: Use 5% non-fat milk or BSA in TBST, with BSA often preferred for phospho-specific detection

• Dilution factors: Most PYCARD antibodies work optimally at 1:500-1:2000 dilution for Western blotting

• Incubation conditions: Primary antibody incubation at 4°C overnight often yields cleaner results than shorter room temperature incubations

• Expected band size: PYCARD typically appears at 21-22 kDa, though the observed molecular weight may differ from calculations due to post-translational modifications

• Membrane selection: PVDF membranes generally provide better protein retention than nitrocellulose for PYCARD detection

Note that mobility rates affecting the observed band size may cause inconsistency with expected size due to factors such as different modified forms of the protein appearing simultaneously on the membrane .

How can specificity of PYCARD antibody binding be validated?

Rigorous validation approaches for PYCARD antibodies include:

• Positive controls: Use cell lysates known to express PYCARD (K562, HeLa, 3T3, 293 cells)

• Genetic knockout controls: Compare results with PYCARD-deficient samples

• Blocking peptide experiments: Pre-incubate antibody with immunizing peptide to confirm specific binding

• Immunoprecipitation followed by mass spectrometry: Confirm target identity

• Multiple antibody validation: Use antibodies recognizing different epitopes of PYCARD

Researchers should be aware that antibodies raised against synthetic peptides derived from the N-terminal region of human ASC may show different binding characteristics than those recognizing full-length protein .

How can PYCARD antibodies be used to investigate inflammasome formation dynamics?

For investigating inflammasome assembly:

• Immunofluorescence microscopy: Track PYCARD redistribution from diffuse cytoplasmic patterns to distinct ASC specks upon inflammasome activation

• Proximity ligation assays: Detect protein-protein interactions between PYCARD and other inflammasome components

• Time-lapse imaging: Monitor the kinetics of ASC speck formation using fluorescently-tagged antibodies

• Co-immunoprecipitation: Identify binding partners during different stages of inflammasome assembly

Cellular localization analysis should note that upstream of caspase activation, PYCARD redistributes from cytoplasm to aggregates, appearing as hollow, perinuclear spherical, ball-like structures .

What strategies address the challenges in detecting specific PYCARD isoforms?

Isoform-specific detection requires:

• Epitope mapping: Select antibodies targeting regions unique to specific isoforms

• Electrophoretic separation: Use high-percentage or gradient gels to resolve closely sized isoforms

• Two-dimensional electrophoresis: Separate isoforms by both isoelectric point and molecular weight

• RT-PCR validation: Confirm isoform expression at mRNA level before protein analysis

• Mass spectrometry: Definitively identify peptides unique to each isoform

When working with PYCARD, be aware that at least three isoforms may be detected by some antibodies, though specific isoform reactivity varies between antibody clones .

How do genetic variants in the PYCARD gene affect antibody binding and experimental outcomes?

Genetic variations in PYCARD can significantly impact research results:

• SNP effects: Single nucleotide polymorphisms, particularly in 3' untranslated regions, can alter expression levels, as demonstrated in mouse models where a single base change (T to A on coding strand) significantly reduced Pycard expression and inflammasome activity

• Epitope alterations: Mutations within antibody binding sites may reduce recognition efficiency

• Expression level variation: Genetic variants can alter transcription rates or mRNA stability

• Post-translational modifications: Genetic variants may create or eliminate modification sites

Research has shown that CRISPR/Cas9 homology directed repair (HDR) gene editing can be used to investigate these effects by changing specific alleles and measuring the impact on inflammasome activation and PYCARD expression .

What are the critical differences between monoclonal and polyclonal PYCARD antibodies?

How do different conjugates affect the performance of PYCARD antibodies?

While specific HRP-conjugated PYCARD antibodies are not directly compared in the search results, general principles apply:

• HRP conjugates: Offer excellent sensitivity for WB, ELISA and IHC with various detection substrates

• Fluorescent conjugates (e.g., Alexa Fluor 488): Provide direct visualization without substrate development, ideal for flow cytometry and IF

• Biotin conjugates: Allow signal amplification through avidin/streptavidin systems

• Unconjugated antibodies: Offer flexibility with secondary detection systems but require additional incubation steps

The selection should be based on the specific application requirements, detection system availability, and desired sensitivity levels .

What are common issues when using PYCARD antibodies and how can they be resolved?

For PYCARD specifically, note that the actual band is not always consistent with expectations due to factors affecting mobility rates in electrophoresis .

How can researchers optimize multiplex detection systems involving PYCARD?

For effective multiplex detection:

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

• Sequential detection: For co-localization studies, perform sequential rather than simultaneous staining

• Fluorophore selection: Choose fluorophores with minimal spectral overlap

• Cross-adsorbed secondaries: Use highly cross-adsorbed secondary antibodies to minimize species cross-reactivity

• Controls: Include single-stain controls to verify specificity in multiplex experiments

These approaches enable simultaneous investigation of PYCARD alongside other inflammasome components or downstream effectors in complex biological systems.