PADI2 Antibody

What criteria should I use to select the appropriate PADI2 antibody for my experiment?

When selecting a PADI2 antibody, consider these critical factors:

• Target species reactivity: Verify documented reactivity with your species of interest. Available PADI2 antibodies show reactivity with human, mouse, rat, rabbit, and pig samples .

• Antibody type: Determine whether polyclonal (e.g., 12110-1-AP) or monoclonal (e.g., 66386-1-Ig) better suits your research needs. Polyclonals offer higher sensitivity but potential batch variation, while monoclonals provide superior specificity and reproducibility .

• Validated applications: Select antibodies validated for your specific application:

  Antibody	Validated Applications	Species Reactivity
  12110-1-AP	WB, IHC, IF/ICC, IF-P, IP, COIP, ELISA	Human, mouse, rat
  66386-1-Ig	WB, IHC, IF-P, ELISA	Human, mouse, rat, rabbit, pig
  AF7257	IHC-P	Human

• Epitope recognition: Check which region of PADI2 the antibody targets, particularly important when studying specific domains or isoforms.

• Validation data: Review published literature citing the antibody to confirm performance in applications similar to yours .

How can I verify the specificity of my PADI2 antibody?

To confirm antibody specificity:

• Positive controls: Use tissues/cells with known PADI2 expression such as:

  • Brain tissue (mouse, rat, human, pig)

  • MCF-7 cells (breast cancer line)

  • Skeletal muscle tissue

  • HeLa cells

• Knockout/knockdown validation: Compare staining between wildtype and PADI2-knockout or PADI2-siRNA samples. Published studies have validated antibodies using PADI2-/- mice and knockdown experiments .

• Molecular weight verification: Confirm band detection at the expected molecular weight (70-75 kDa). Some PADI2 antibodies may also detect a secondary band at ~50 kDa representing an isoform .

• Pre-adsorption control: Pre-incubate antibody with recombinant PADI2 protein to confirm signal elimination.

• Multiple antibody validation: Use two different antibodies targeting distinct PADI2 epitopes to confirm consistent localization patterns.

What are the recommended storage conditions and handling practices for PADI2 antibodies?

For optimal antibody performance and shelf life:

• Storage temperature: Most PADI2 antibodies should be stored at -20°C. Avoid repeated freeze-thaw cycles .

• Storage buffer: Typically supplied in PBS with 0.02% sodium azide and 50% glycerol, pH 7.3 .

• Aliquoting: For larger volumes, aliquoting is recommended to avoid repeated freeze-thaw cycles, though some suppliers note it's unnecessary for -20°C storage .

• Stability: Most PADI2 antibodies remain stable for one year after shipment when stored properly .

• Working solution: For diluted working solutions, store at 4°C and use within 1 month. For longer storage, return to -20°C .

• Light sensitivity: For fluorophore-conjugated antibodies (e.g., CL488-66386), avoid exposure to light .

What are the optimal dilution ranges for PADI2 antibodies across different applications?

Recommended dilutions vary by application and specific antibody:

Important considerations:

• Sample-dependent optimization is recommended

• Titrate each antibody in your specific system

• Higher concentrations may be needed for weakly expressed samples

• Lower concentrations for strongly expressed samples to reduce background

What are the recommended protocols for immunohistochemistry (IHC) with PADI2 antibodies?

For optimal IHC results with PADI2 antibodies:

• Fixation: Use 10% neutral buffered formalin or 4% paraformaldehyde.

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Blocking: Use appropriate blocking buffer (e.g., 5% BSA or normal serum) for 1 hour at room temperature.

• Primary antibody incubation:

  • Dilution: 1:50-1:500 (antibody-dependent)

  • Incubation: Overnight at 4°C (recommended for most PADI2 antibodies)

• Detection system: For mouse-origin antibodies like 66386-1-Ig, use anti-mouse HRP or fluorophore-conjugated secondary antibodies. For rabbit-origin antibodies like 12110-1-AP, use anti-rabbit detection systems .

• Positive control tissues: Human breast cancer tissue shows consistent PADI2 expression, making it an ideal positive control .

• Special application: For Alzheimer's disease studies, human brain (cortex) tissue works well with the sheep anti-human PADI2 antibody (AF7257) at 3 μg/mL overnight at 4°C, using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit .

How should I optimize Western blotting protocols for PADI2 detection?

For optimal Western blot detection of PADI2:

• Protein extraction: For general applications, use RIPA buffer with protease inhibitors. For histone citrullination studies, specialized histone extraction protocols are recommended .

• Gel conditions:

  • 10% Bis-tris gel under MOPS buffer system

  • Run at 200V for approximately 50 minutes

• Transfer conditions:

  • Nitrocellulose membrane preferred

  • Transfer at 30V for 70 minutes

• Blocking:

  • 5% Bovine Serum Albumin in TBS-T for 1 hour at room temperature

• Primary antibody incubation:

  • Dilution range: 1:500-1:6000 (antibody-dependent)

  • Incubate overnight at 4°C

• Expected band sizes:

  • Primary band: 70-75 kDa

  • Secondary band (isoform): ~50 kDa in some samples

• Positive controls: MCF-7 cells, brain tissue (human, mouse, rat), and skeletal muscle tissue consistently show strong PADI2 expression .

How can I effectively design experiments to study PADI2's role in breast cancer?

Based on published research on PADI2 in breast cancer:

• Cell line selection:

  • Use MCF10AT model to study progression (normal to malignant)

  • MCF10DCIS cells show peak PADI2 expression (modeling human comedo-DCIS lesions)

  • MCF-7 cells are positive for PADI2 expression

• Expression analysis:

  • Correlate PADI2 levels with HER2/ERBB2 status

  • PADI2 is significantly upregulated in luminal breast cancer cell lines (p = 2.2 × 10^-6)

• Functional studies:

  • Use PADI inhibitors like Cl-amidine to assess growth effects

  • Examine both 2D-monolayers and 3D-spheroids for comprehensive analysis

  • Assess xenograft tumor growth in nude mice models

• Molecular mechanisms:

  • Analyze cell cycle gene expression changes (p21, GADD45α, Ki67)

  • Examine histone citrullination as a potential epigenetic mechanism

• Clinical correlation:

  • Compare PADI2 expression in normal vs. tumor tissues

  • Correlate with HER2/ERBB2 status in clinical samples

This experimental design follows the approach used in studies that identified PADI2 as a potential breast cancer biomarker, particularly in HER2/ERBB2+ tumors .

What controls should I include when studying PADI2-mediated citrullination?

When studying PADI2-mediated citrullination, include these essential controls:

• Positive controls for PADI2 activity:

  • Known PADI2 substrates: vimentin, actin, myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and histones

  • Anti-citrullinated protein antibodies (e.g., anti-H3Cit2,8,17) to detect citrullination

• Negative controls:

  • PADI2 knockout/knockdown samples

  • PADI inhibitors (e.g., Cl-amidine) treatment

  • Calcium chelation (PADI2 activity is Ca²⁺-dependent)

• Specificity controls:

  • Non-citrullinated target proteins

  • Other PADI family members' activity

  • Site-directed mutagenesis of target arginine residues

• Technical controls:

  • Western blot detection of both PADI2 and citrullinated proteins

  • Immunoprecipitation of PADI2 followed by activity assays

  • Mass spectrometry to confirm citrullination sites

For histone citrullination studies specifically, compare wildtype and PADI2-/- samples using anti-H3Cit2,8,17 antibodies as demonstrated in published research .

How can I distinguish between different PADI isoforms in my experiments?

To distinguish between the five known PADI isozymes (PADI1-4 and PADI6):

• Antibody selection:

  • Use highly specific antibodies validated against multiple PADI family members

  • Verify specificity using knockout/knockdown models

  • Consider epitopes unique to PADI2 (sequence alignment analysis can help identify these regions)

• Expression pattern analysis:

  • PADI2 is the most widely expressed, found in muscle, brain, colon, breast, macrophages, spleen, and spinal cord

  • PADI4 is predominantly expressed in granulocytes and macrophages

  • PADI1 and PADI3 are mainly in epidermis and hair follicles

  • PADI6 is primarily in oocytes and early embryos

• Molecular analysis:

  • RT-qPCR with isoform-specific primers

  • Western blotting comparing molecular weights (PADI2: 75 kDa)

  • Mass spectrometry for protein identification

• Functional differentiation:

  • PADI2 primarily citrullinates structural proteins and histones

  • PADI4 is known for nuclear localization and histone modification

  • PADI6 functions during zygotic genome activation

• Knockout models:

  • Generate or obtain isoform-specific knockout models

  • Verify antibody specificity and functional effects in these models

How can I investigate PADI2's role in epigenetic regulation through histone citrullination?

To study PADI2's epigenetic functions:

• Chromatin immunoprecipitation (ChIP):

  • Use anti-PADI2 antibodies to identify genomic regions where PADI2 is bound

  • Follow with ChIP-seq for genome-wide analysis

  • Correlate with histone citrullination patterns

• Histone citrullination detection:

  • Western blotting using anti-H3Cit2,8,17 antibodies

  • Protocol: Follow manufacturer's histone extraction guidelines (Abcam)

  • Compare wildtype vs. PADI2-/- or PADI2-knockdown samples

• Gene expression correlation:

  • RNA-seq following PADI2 knockdown/knockout

  • Focus on genes involved in cell cycle regulation (p21, GADD45α, Ki67)

  • Analyze PADI2's effect on SOX9 target genes (Ptgds, Cyp26b1)

• Mechanistic studies:

  • Co-immunoprecipitation to identify PADI2 interaction partners

  • siRNA knockdown of PADI2 combined with transcriptional reporter assays

  • PADI inhibitors (Cl-amidine) treatment to assess functional outcomes

• Tissue-specific analysis:

  • For breast cancer: MCF-7 cells show high PADI2 expression

  • For neurological studies: brain tissue samples

  • For testicular development: Sertoli cells (SOX9-regulated)

Research has shown that PADI2 can modify SOX9 transcriptional activity, suggesting a role in epigenetic regulation of gene expression .

What are the best experimental approaches to study PADI2's involvement in neurodegenerative diseases?

For investigating PADI2's role in neurodegenerative conditions:

• Tissue and model selection:

  • Human Alzheimer's brain (cortex) samples

  • Mouse models of neurodegeneration

  • Neuronal and glial cell cultures

• PADI2 localization in neural tissue:

  • Immunohistochemistry using anti-PADI2 antibodies

  • AF7257 antibody has been validated for human Alzheimer's brain at 3 μg/mL

  • Co-localization with neuronal and glial markers

• Target protein citrullination:

  • Focus on known neural substrates: myelin basic protein (MBP), glial fibrillary acidic protein (GFAP)

  • Western blot and immunohistochemistry to detect citrullinated proteins

  • Compare citrullination patterns between normal and diseased samples

• Functional studies:

  • PADI inhibitors in neurodegeneration models

  • PADI2 knockout/knockdown in neuronal/glial cultures

  • Assessment of neuroinflammatory markers and pathways

• Clinical correlation:

  • PADI2 expression/activity correlation with disease progression

  • Citrullinated protein levels in CSF as potential biomarkers

  • Genetic association studies (PADI2 polymorphisms)

Research has established that excessive PAD-mediated deimination of MBP contributes to multiple sclerosis progression, while elevated levels of citrullinated GFAP and vimentin have been found in Alzheimer's disease patients' brains .

How can I design experiments to investigate the transcriptional regulation of PADI2?

To study factors controlling PADI2 expression:

• Promoter analysis:

  • Clone PADI2 5'-UTR and intronic fragments into luciferase reporter vectors

  • Test constructs with mutations in putative transcription factor binding sites

  • Research has identified SOX9 response elements in the PADI2 promoter

• Transcription factor binding:

  • ChIP assays to detect binding of suspected transcription factors (e.g., SOX9)

  • EMSA to confirm direct binding to PADI2 regulatory regions

  • Co-transfection experiments with transcription factors and PADI2 reporters

• Expression studies:

  • RT-qPCR analysis of PADI2 in different cell types and conditions

  • Western blot confirmation of protein expression

  • Correlate with developmental stages or disease progression

• Functional analysis:

  • siRNA knockdown of suspected regulators (e.g., SOX9)

  • Measure effects on PADI2 expression

  • Assess downstream functional consequences

• Cell-type specificity:

  • In testicular development: PADI2 is specifically expressed in Sertoli cells

  • In breast cancer: expression correlates with luminal subtype and HER2 status

  • Design studies to understand tissue-specific regulation mechanisms

Research has demonstrated that SOX9 is a transcriptional activator of PADI2, particularly in Sertoli cells during testicular development .

What could cause multiple bands when detecting PADI2 by Western blotting?

Multiple bands in PADI2 Western blots can result from:

• Known isoforms:

  • Primary band: 70-75 kDa (full-length PADI2)

  • Secondary band: ~50 kDa (reported isoform)

  • These patterns are documented in product specifications

• Post-translational modifications:

  • Phosphorylation, glycosylation, or other modifications

  • Can cause mobility shifts or appearance of additional bands

  • Compare with phosphatase-treated samples to confirm

• Degradation products:

  • Improper sample handling or storage

  • Insufficient protease inhibitors during extraction

  • Avoid repeated freeze-thaw cycles of protein lysates

• Non-specific binding:

  • Try higher antibody dilutions (1:2000-1:6000)

  • More stringent washing steps

  • Different blocking agents (milk vs. BSA)

• Cross-reactivity:

  • With other PADI family members

  • Verify with PADI2 knockout/knockdown samples

  • Try alternative validated antibodies targeting different epitopes

For consistent results, use freshly prepared samples, optimize protein loading, and include appropriate positive controls like MCF-7 cells or brain tissue .

How can I resolve weak or inconsistent PADI2 staining in immunohistochemistry?

To improve PADI2 immunostaining results:

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Extend retrieval time for challenging samples

• Fixation considerations:

  • Overfixation can mask epitopes

  • Standardize fixation times (10% NBF, 24-48 hours recommended)

  • Consider testing different fixatives if consistent issues occur

• Antibody concentration:

  • For weak signal: Try higher antibody concentration (1:50-1:100)

  • For high background: More dilute antibody (1:200-1:500)

  • Extend incubation time (overnight at 4°C recommended)

• Detection system:

  • Consider amplification methods (e.g., tyramide signal amplification)

  • Use polymer-based detection for improved sensitivity

  • Match detection system to primary antibody host species

• Positive controls:

  • Include validated positive control tissues

  • Human breast cancer tissue and brain tissue consistently show PADI2 expression

  • Process controls alongside test samples to ensure protocol consistency

• Antibody selection:

  • If one antibody fails, try an alternative validated PADI2 antibody

  • Consider both polyclonal (e.g., 12110-1-AP) and monoclonal (e.g., 66386-1-Ig) options

How should I interpret contradictory PADI2 expression data between different experimental methods?

When facing discrepancies in PADI2 data across methods:

• Consider method-specific limitations:

  • IHC/IF shows localization but has semi-quantitative results

  • Western blot provides size information but may miss localized changes

  • qPCR measures mRNA but not protein translation or stability

• Expression vs. activity discrepancies:

  • PADI2 protein presence doesn't guarantee enzymatic activity

  • Ca²⁺-dependency means cellular calcium levels affect function

  • Measure both PADI2 expression and citrullinated target proteins

• Tissue/cell heterogeneity:

  • Whole tissue lysates may mask cell-specific expression changes

  • PADI2 expression is cell-type specific (e.g., Sertoli cells in testes)

  • Consider single-cell approaches or cell sorting

• Antibody-specific factors:

  • Different antibodies recognize different epitopes

  • Some epitopes may be masked in certain experimental conditions

  • Use multiple validated antibodies targeting different regions

• Functional validation:

  • PADI2 knockout/knockdown to confirm specificity

  • Enzymatic activity assays to confirm functional relevance

  • Substrate citrullination as a functional readout