paxip1 Antibody

What is PAXIP1 and what are its primary functions in cellular processes?

PAXIP1 (PAX interacting protein 1) is a nuclear protein with six BRCT (breast cancer carboxy-terminal) domains, with a molecular weight of approximately 121.3 kilodaltons . PAXIP1 functions in multiple cellular processes:

• DNA damage response and repair: Forms complexes with TP53BP1 to regulate ATM association and promotes PCNA ubiquitination following UV irradiation

• Epigenetic regulation: Associates with MLL3/MLL4-containing histone H3K4 methyltransferase complexes

• Immunological processes: Involved in immunoglobulin class switching in activated B-cells through H3K4 trimethylation and V(D)J recombination

• 3D genome organization: Critical for maintaining genome architecture and facilitating promoter/enhancer contacts during transcription

What experimental applications are PAXIP1 antibodies optimized for?

PAXIP1 antibodies have been validated for multiple research applications:

• Western blot (WB): For protein expression quantification

• Immunohistochemistry (IHC): For tissue localization studies

• Immunofluorescence (ICC-IF): For cellular localization

• Chromatin immunoprecipitation (ChIP): For studying DNA-protein interactions

• Immunoprecipitation: For protein complex isolation and interaction studies

The choice of application should align with your specific research question. For example, ChIP applications are critical when investigating PAXIP1's role in transcriptional regulation, while co-immunoprecipitation is essential for characterizing protein-protein interactions.

How should I validate PAXIP1 antibody specificity for my experimental system?

Methodological approach to validation:

• Positive and negative controls:

  • Use cell lines with documented high PAXIP1 expression (e.g., certain OC cell lines) as positive controls

  • Compare against PAXIP1 knockout models or PAXIP1-depleted cell lines as negative controls

• Multiple detection methods:

  • Cross-validate protein detection using at least two applications (e.g., WB and IF)

  • Confirm specificity using siRNA knockdown or CRISPR knockout models

• Experimental validation:

  • For ChIP applications, confirm enrichment at known binding sites (e.g., GR-bound sites)

  • For co-IP experiments, verify interactions with known binding partners like PAGR1

How can I effectively use PAXIP1 antibodies to investigate DNA damage response pathways?

Methodological approach:

• Co-immunoprecipitation protocols:

  • Use CHAPS lysis buffer (0.5% CHAPS, 150 mM NaCl, 10 mM Hepes, pH 7.4)

  • Incubate 500μg whole cell lysate with 5μg anti-PAXIP1 antibody

  • Form immune complexes for 1 hour at 4°C

  • Add 20μl protein A/G Sepharose beads and incubate overnight at 4°C with rotation

  • Wash three times with lysis buffer, boil in Laemmli buffer for 5 minutes

  • Analyze by Western blotting for DNA damage response components (e.g., BARD1, WEE1)

• ChIP-qPCR for DNA damage sites:

  • Target known DNA damage sites where PAXIP1 interacts with TP53BP1

  • Use 5μg of PAXIP1 antibody per immunoprecipitation

  • Include controls for non-damage sites to establish specificity

• Functional verification experiments:

  • Combine with AZD1775 (WEE1 inhibitor) treatment to assess PAXIP1's role in DNA damage response

  • Monitor caspase 3-mediated apoptosis as a readout of the pathway's functionality

What methodological approaches can resolve contradictions in PAXIP1 functional studies?

The literature shows some contradictory results about PAXIP1's roles. Here's a methodological framework to address these contradictions:

• Cell-type specific function analysis:

  • Compare PAXIP1 function across multiple cell types (e.g., T cells, B cells, cancer cell lines)

  • The data indicates different phenotypes in different contexts - e.g., T cell development vs. cancer cell responses

• Complex-specific function discrimination:

  • Design experiments to differentiate between PAXIP1's roles in:

    • MLL3/MLL4-containing histone methyltransferase complexes

    • DNA damage repair complexes with PAGR1

    • Cohesin-associated genome organization functions

  • Use sequential immunoprecipitation or proximity ligation assays to isolate specific complexes

• Domain-specific mutational analysis:

  • Generate constructs expressing PAXIP1 with mutations in specific BRCT domains

  • For example, the tBRCT C2 domain interacts with WEE1

  • Perform complementation studies in PAXIP1 knockout cells

• Context-dependent regulation assessment:

  • Evaluate PAXIP1 function under different cellular stresses (DNA damage, hormone stimulation)

  • PAXIP1 shows differential activity during glucocorticoid treatment vs. cisplatin exposure

How can I use PAXIP1 antibodies to study 3D genome architecture and enhancer-promoter interactions?

Advanced methodological approaches:

• ChIP-seq for cohesin and PAXIP1 co-occupancy:

  • Perform sequential or parallel ChIP for PAXIP1 and cohesin components (RAD21, SMC1)

  • Focus analysis on regions showing hormone-dependent binding (e.g., glucocorticoid response elements)

  • 5μg of RAD21, PAXIP1, and H3K4me1 antibodies are recommended per ChIP

• 4C-seq with PAXIP1 modulation:

  • Use promoter regions of PAXIP1-regulated genes (e.g., FKBP5) as viewpoints

  • Compare wild-type vs. PAXIP1-depleted cells to assess changes in chromatin interactions

  • Study both basal and stimulus-induced (e.g., glucocorticoid) interactions

• Live-cell imaging with FRAP:

  • Tag cohesin components (e.g., SMC1-EGFP) in control and PAXIP1-depleted cells

  • Measure recovery after photobleaching to assess cohesin mobility and chromatin stability

  • Calculate immobile fractions to determine the impact of PAXIP1 on cohesin dynamics

• Correlation with epigenetic marks:

  • Perform parallel ChIP-seq for PAXIP1 and histone modifications (e.g., H3K4me3, H3K4me1)

  • Analyze how PAXIP1 loss affects these marks at enhancers and promoters

What experimental designs best characterize the relationship between PAXIP1 and its antisense transcript PAXIP1-AS1?

Recent studies have identified PAXIP1-AS1 as a functional long non-coding RNA with roles distinct from PAXIP1 protein. Here's a methodological approach to study their relationship:

• Expression correlation analysis:

  • Quantify PAXIP1 protein and PAXIP1-AS1 transcript levels across tissue samples

  • In gastric cancer, PAXIP1-AS1 expression negatively correlates with HOXD9 expression

  • In ovarian cancer, low PAXIP1-AS1 expression correlates with poor prognosis

• Subcellular localization studies:

  • Perform cellular fractionation followed by qPCR to determine PAXIP1-AS1 localization

  • Use immunofluorescence with PAXIP1 antibodies to compare protein localization

  • PAXIP1-AS1 shows higher expression in cytoplasm than nucleus in GC cells

• Functional interaction experiments:

  • Design knockdown/overexpression experiments for both PAXIP1 and PAXIP1-AS1

  • For PAXIP1-AS1 studies:

    • Establish stable transfectants with PAXIP1-AS1 overexpression

    • Use siRNA for knockdown studies

    • Measure effects on proliferation using EdU incorporation assays

• Regulatory mechanism investigation:

  • Perform ChIP analysis to identify transcription factors regulating PAXIP1-AS1

  • Use luciferase reporter assays to validate transcription factor binding sites

  • HOXD9 has been shown to bind PAXIP1-AS1 promoter at position -1503 to -1513

How should I design experiments to evaluate PAXIP1's role in cancer progression?

Evidence suggests PAXIP1 and PAXIP1-AS1 play roles in cancer development. Here's a methodological framework:

What are the methodological considerations for studying PAXIP1 in immune system development?

PAXIP1 plays critical roles in immune system processes. Here's how to investigate these functions:

• T cell development studies:

  • Generate conditional knockout models (e.g., PAXIP1 × Lck Cre mice)

  • Analyze thymocyte development:

    • Flow cytometry for CD4/CD8 populations

    • TCRβ, CD69, Qa2, and HSA markers for maturation

  • Examine peripheral T cell populations in lymph nodes, spleen, and blood

• V(D)J recombination analysis:

  • Study TCR diversity through PCR-based Jα usage analysis

  • Use RT-PCR with TRAV12 (Vα8) family primers and Cα primers

  • Analyze results through Southern blotting with Jα-specific oligonucleotide probes

• Histone modification assessment:

  • Investigate H3K4me3 modifications at Jα elements

  • Compare wild-type versus PAXIP1-deficient models

  • Analyze germline transcripts at accessible Jα elements

• Class switch recombination studies:

  • Examine IgG3 and IgG1 class switching in B cells

  • Analyze AID-dependent DNA breaks through γ-H2AX foci formation

  • Study PAXIP1's role in repair of AID-dependent DSBs

What are the optimal conditions for co-immunoprecipitation experiments with PAXIP1 antibodies?

Based on published protocols:

• Buffer composition and lysis conditions:

  • CHAPS lysis buffer (0.5% CHAPS, 150 mM NaCl, 10 mM Hepes, pH 7.4)

  • 500μg of whole cell lysate per immunoprecipitation

  • 5μg of anti-PAXIP1 antibody per reaction

• Interaction formation parameters:

  • Form immune complexes for 1 hour at 4°C

  • Add 20μl of protein A/G Sepharose beads

  • Incubate overnight with rotation at 4°C

• Washing and elution:

  • Wash beads three times with lysis buffer

  • Elute by boiling in Laemmli buffer for 5 minutes

  • Analyze by Western blotting

• Controls and validation:

  • Include negative controls (IgG)

  • Verify known interactions (PAGR1, BARD1, WEE1)

  • Confirm through reverse immunoprecipitation

How can I optimize ChIP protocols for PAXIP1 to study its genome-wide binding patterns?

Detailed methodological approach:

• Crosslinking and chromatin preparation:

  • Standard formaldehyde crosslinking (1% for 10 minutes)

  • Sonication to achieve fragments of 200-500bp

• Antibody selection and amount:

  • Use 5μg of PAXIP1 antibody per ChIP reaction

  • Include controls:

    • Non-specific IgG

    • Input chromatin

• Washing and elution conditions:

  • Follow standard ChIP protocols with sequential washes of increasing stringency

  • Reverse crosslinks at 65°C overnight

• Analysis approaches:

  • ChIP-qPCR for targeted regions (GR binding sites, promoters)

  • ChIP-seq for genome-wide binding patterns

  • Bioinformatic analysis focusing on:

    • Introns and distal intergenic regions (major sites of GR-PAXIP1-RAD21 co-occupancy)

    • Motif analysis for glucocorticoid response elements, JUN, and forkhead transcription factors (FOX)

• Integration with other datasets:

  • Combine with RNA-seq to correlate binding with gene expression

  • Integrate with histone modification data (H3K4me1, H3K4me3)

  • Compare with cohesin binding patterns (RAD21, SMC1)