PA2G4 Antibody

What is PA2G4 and why is it significant in cancer research?

PA2G4 (EBP1) was first identified as an ErbB3 binding protein and is a 38 kDa protein widely expressed in cultured cells and tissues. It contains several functional domains including a nuclear localization sequence (NLS), LxxLL and LxCxE motifs, suggesting its involvement in cell signaling pathways . The protein is particularly significant in cancer research due to its interaction with MYCN, where it functions as a cofactor that stabilizes MYCN and prevents its degradation by the Ubiquitin Proteasome System . This interaction has been identified as a potential therapeutic target, particularly in MYCN-amplified neuroblastoma treatments .

What are the key differences between monoclonal and polyclonal PA2G4 antibodies in research applications?

Monoclonal PA2G4 antibodies (such as 66055-1-Ig) offer high specificity but recognize a single epitope, while polyclonal antibodies (such as 15348-1-AP and PAL980Hu01) recognize multiple epitopes, potentially providing stronger signals in certain applications . The choice between them depends on experimental needs:

For experimental reproducibility, monoclonal antibodies are often preferred, while polyclonal antibodies may be advantageous for detecting proteins with post-translational modifications or conformational changes .

How consistent are the observed molecular weights of PA2G4 across different experimental systems?

There's a notable discrepancy between the calculated and observed molecular weights of PA2G4. While the calculated molecular weight is 44 kDa, researchers have observed molecular weights of 38-42 kDa and 48 kDa in experimental settings . This variation may reflect:

• Post-translational modifications

• Different protein isoforms

• Tissue or cell-specific processing

• Variation in experimental conditions including gel composition

Researchers should anticipate detecting bands at these molecular weights when performing Western blot analysis and should be prepared to validate bands using appropriate controls such as recombinant PA2G4 protein or knockout/knockdown cells .

What are the optimal antibody dilutions for different applications, and how should they be determined?

The optimal dilution varies significantly based on application type and specific experimental conditions:

Methodologically, optimal dilutions should be determined through titration experiments for each specific sample type and experimental system. Begin with the manufacturer's recommended range and perform a dilution series, selecting the concentration that provides maximum specific signal with minimal background .

What antigen retrieval methods are most effective for PA2G4 immunohistochemistry in different tissue types?

For optimal PA2G4 detection in tissue samples, two main antigen retrieval methods have shown effectiveness:

• Primary recommendation: TE buffer pH 9.0

• Alternative method: Citrate buffer pH 6.0

These recommendations apply particularly to human breast cancer tissue samples, but researchers working with other tissue types should conduct comparative studies . The efficacy of antigen retrieval can vary depending on:

• Fixation method and duration

• Tissue type and processing

• Antibody clone used

• Target protein localization

A methodological approach for optimizing antigen retrieval would include testing both buffers in parallel sections, varying retrieval times (10-30 minutes), and assessing both signal intensity and background levels quantitatively .

How should researchers design validation experiments for PA2G4 antibody specificity?

A comprehensive antibody validation strategy should include:

• Positive controls: Use cell lines with confirmed PA2G4 expression like HeLa, HEK-293, PC-3, Jurkat, K-562 cells

• Negative controls: Include:

  • Primary antibody omission

  • Isotype controls

  • PA2G4 knockout/knockdown cells via CRISPR or siRNA

• Peptide competition assay: Pre-incubate antibody with purified PA2G4 protein or immunogenic peptide

• Cross-reactivity assessment: Test in tissue known to lack PA2G4 expression

• Multiple antibody validation: Compare results using different PA2G4 antibody clones recognizing distinct epitopes

Importantly, validation should be performed for each specific application (WB, IHC, IF, IP) as antibody performance can vary significantly between applications .

How can PA2G4 antibodies be utilized to investigate the PA2G4-MYCN interaction in neuroblastoma research?

Recent research reveals PA2G4 as a cofactor of MYCN that stabilizes MYCN and prevents its degradation by the Ubiquitin Proteasome System . To investigate this interaction:

• Co-immunoprecipitation (Co-IP): Use PA2G4 antibodies for IP followed by MYCN detection via Western blot. When selecting antibodies for this application, consider using rabbit anti-PA2G4 for IP and mouse anti-MYCN for detection to avoid heavy chain interference in Western blotting .

• Proximity Ligation Assay (PLA): Detect in situ protein-protein interactions with spatial resolution using specific PA2G4 and MYCN antibodies.

• Compound screening: Evaluate potential disruptors of PA2G4-MYCN interaction using co-IP after treatment with compounds of interest. WS6 analogues like compounds #5333 and #5338 have shown promise in disrupting this interaction .

• Expression correlation studies: Use IHC with PA2G4 and MYCN antibodies on sequential tissue sections to correlate expression patterns in clinical samples.

The technical challenge in these applications is selecting antibodies that don't compete for the same binding region involved in the PA2G4-MYCN interaction .

What methods are recommended for studying PA2G4 in relation to cancer mechanisms and therapeutic targets?

PA2G4 appears to play a significant role in cancer through its interactions with oncoproteins like MYCN, suggesting several investigative approaches:

• Protein complex analysis: Use PA2G4 antibodies in IP-mass spectrometry approaches to identify protein complexes across different cancer types. This can reveal tissue-specific interaction partners.

• Functional domain mapping: Combine PA2G4 antibodies recognizing different epitopes with mutational analysis to determine which domains are critical for specific protein-protein interactions.

• Compound efficacy assessment: Use PA2G4 antibodies to monitor protein degradation or stabilization following treatment with compounds targeting PA2G4-protein interactions. Note that compounds like #5333 and #5338 can decrease both MYCN and PA2G4 protein levels, suggesting complex mechanisms .

• Transcriptional regulation studies: Use chromatin immunoprecipitation (ChIP) with PA2G4 antibodies to identify genomic regions where PA2G4 participates in transcriptional regulation, particularly in relation to GATA2, which appears to be regulated by MYCN/PA2G4 .

• Xenograft studies: Use IHC with PA2G4 antibodies to assess protein expression and localization in tumor xenograft models before and after treatment with potential therapeutic compounds .

How can researchers interpret contradictory results regarding PA2G4 stability following treatment with small molecule inhibitors?

An interesting paradox observed in recent research is that compounds like WS6 analogues (#5333 and #5338) that bind to PA2G4 appear to decrease its protein levels, despite differential scanning fluorimetry (DSF) data suggesting a stabilizing effect (increased melting temperature) . To resolve this contradiction, researchers should consider:

• Mechanistic investigations:

  • Determine if the compound triggers conformational changes that expose degradation signals

  • Investigate whether binding triggers autoregulatory feedback loops affecting PA2G4 transcription

  • Assess if compound binding affects post-translational modifications that influence stability

• Time-course experiments: Monitor PA2G4 levels at multiple time points after treatment to distinguish between immediate stabilization and subsequent degradation effects.

• Proteasome inhibitor co-treatment: Determine if proteasome inhibitors prevent the observed decrease in PA2G4 levels, indicating a degradation-dependent mechanism.

• Domain-specific antibodies: Use antibodies recognizing different epitopes to determine if the apparent decrease might be due to epitope masking rather than actual protein reduction .

What are the most common sources of false positives/negatives when using PA2G4 antibodies, and how can they be addressed?

Several technical issues can lead to unreliable results when using PA2G4 antibodies:

To systematically address these issues, implement a standardized validation protocol for each new lot of antibody and cell/tissue type, including appropriate positive and negative controls .

What are the critical storage and handling considerations to maintain PA2G4 antibody performance over time?

Proper antibody handling significantly impacts experimental reproducibility:

• Storage conditions:

  • Store at -20°C for long-term storage

  • Antibodies are stable for one year after shipment under proper conditions

  • Aliquoting is not necessary for -20°C storage according to manufacturers, but is recommended for frequent use to avoid freeze-thaw cycles

• Buffer composition:

  • Most PA2G4 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Some preparations (20μL sizes) contain 0.1% BSA as a stabilizer

• Stability testing:

  • Thermal stability can be assessed by accelerated thermal degradation test (37°C for 48h)

  • High-quality antibodies should show less than 5% loss rate under these conditions

• Working solution handling:

  • Prepare fresh dilutions for each experiment

  • Keep on ice during experiments

  • Avoid repeated freeze-thaw cycles of diluted antibody

• Quality control:

  • Include a consistent positive control in each experiment to monitor antibody performance over time

  • Document lot numbers and reactivity patterns for long-term studies

How should researchers optimize fixation protocols for PA2G4 detection in different sample types?

Fixation conditions significantly impact PA2G4 epitope accessibility and detection sensitivity:

• Cell lines (IF/ICC):

  • For HeLa and HepG2 cells, 4% paraformaldehyde (15-20 minutes at room temperature) preserves PA2G4 epitopes effectively

  • Avoid over-fixation which can mask epitopes

  • Methanol fixation may be preferable for nuclear epitope detection

• Tissue samples (IHC):

  • Formalin-fixed paraffin-embedded (FFPE) tissues require appropriate antigen retrieval

  • For human breast cancer, lung cancer, glioma, and pancreatic cancer tissues, successful detection has been reported using:

    • TE buffer pH 9.0 (primary recommendation)

    • Citrate buffer pH 6.0 (alternative method)

• Optimization approach:

  • For new tissue types, conduct parallel processing with multiple fixation protocols

  • Compare 10% neutral buffered formalin, 4% paraformaldehyde, and Bouin's fixative

  • Vary fixation durations (6-24 hours) to determine optimal conditions

  • Quantify signal-to-noise ratio for each condition to identify optimal protocol

How is PA2G4 antibody research contributing to new therapeutic approaches for MYCN-amplified neuroblastoma?

PA2G4 antibodies have facilitated several critical discoveries regarding the PA2G4-MYCN protein-protein interaction, opening new therapeutic avenues:

• Target validation: PA2G4 antibodies have established PA2G4 as a cofactor for MYCN that prevents its degradation, validating this interaction as a therapeutic target .

• Compound screening: PA2G4 antibodies enable screening assays to identify compounds that disrupt the PA2G4-MYCN interaction. WS6 analogues like compounds #5333 and #5338 have shown promising activity with IC50 values of 23.3-30 μM in neuroblastoma cell lines .

• Mechanism elucidation: Research using these antibodies has revealed that disrupting PA2G4-MYCN interaction leads to decreased colony formation and cell viability in neuroblastoma cells, suggesting a viable therapeutic approach .

• Biomarker development: PA2G4 antibodies can potentially be used to develop immunohistochemical assays that assess PA2G4 expression and localization as predictive biomarkers for response to therapies targeting this interaction .

• Combination therapy strategies: Studies suggest that GATA2 may be regulated by MYCN/PA2G4, and GATA2 knockdown sensitizes cells to compounds disrupting the PA2G4-MYCN interaction, pointing to potential combination approaches .

Future directions include developing antibodies specifically targeting the PA2G4-MYCN interaction interface and improving compound potency, as current IC50 values (23-30 μM) are likely too high for clinical translation without further optimization .

What new insights are emerging about PA2G4 functional domains and their roles in different cellular contexts?

PA2G4 (EBP1) contains several functional domains including a nuclear localization sequence (NLS), LxxLL and LxCxE motifs, suggesting complex roles in cellular processes . Ongoing research using domain-specific antibodies is revealing:

• Subcellular localization patterns: PA2G4 has been detected in multiple cellular compartments, suggesting context-dependent functions. Immunofluorescence studies with PA2G4 antibodies show distinct localization patterns in different cell types .

• Interaction networks: Beyond MYCN, PA2G4 appears to participate in multiple protein complexes, with different domains mediating specific interactions. Recent work suggests that these interaction networks may vary between normal and cancer cells .

• Functional consequences: Disrupting specific PA2G4 domains or interactions can have differential effects on cell proliferation, apoptosis, and gene expression, suggesting potential for selective therapeutic targeting .

• Tissue-specific roles: IHC studies with PA2G4 antibodies have demonstrated variable expression and localization patterns across cancer types, including breast cancer, lung cancer, glioma, and pancreatic cancer, suggesting context-dependent functions .

Future research directions include developing antibodies that specifically recognize different PA2G4 isoforms or post-translationally modified forms to dissect their distinct functional roles across cell types and disease states .

How can researchers address the limitations of current PA2G4 antibodies and develop next-generation reagents for emerging applications?

Despite their utility, current PA2G4 antibodies have limitations that future development should address:

• Epitope-specific antibodies: Developing antibodies targeting specific functional domains would enable more precise studies of PA2G4 interactions and conformational changes. Research suggests PA2G4 undergoes significant conformational changes when bound to interacting partners or compounds .

• Isoform-specific detection: Current antibodies may not discriminate between potential PA2G4 isoforms or post-translationally modified variants. Mass spectrometry-guided epitope selection could enable development of modification-specific antibodies .

• Enhanced sensitivity: Improving detection limits would benefit studies in samples with low PA2G4 expression. Signal amplification approaches and high-affinity recombinant antibody formats could address this need.

• Multiplex compatibility: Developing PA2G4 antibodies compatible with multiplex immunofluorescence or mass cytometry would enable simultaneous analysis of PA2G4 with interacting partners like MYCN in single cells .

• Intrabodies and proximity labeling applications: Engineering PA2G4 antibody fragments for intracellular expression (intrabodies) or fusion to proximity labeling enzymes would enable novel applications for mapping PA2G4 interactions in living cells.