POGZ Antibody

What are the validated applications for POGZ antibodies?

POGZ antibodies have been rigorously validated for multiple applications in research settings. Commercial antibodies like 30106-1-AP have demonstrated efficacy in Western blotting (WB), immunohistochemistry (IHC), immunofluorescence/immunocytochemistry (IF/ICC), immunoprecipitation (IP), and ELISA applications . Each application requires specific optimization for best results. For example, Western blotting typically employs dilutions of 1:1000-1:6000, while IHC applications generally use 1:50-1:500 dilutions . For PACO29046 antibody, ELISA applications recommend dilutions of 1:2000-1:10000, while IHC applications suggest 1:20-1:200 .

How should POGZ antibodies be stored to maintain optimal activity?

Proper storage is crucial for maintaining antibody integrity and activity. POGZ antibodies are typically supplied in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . These antibodies should be stored at -20°C, where they remain stable for approximately one year after shipment . Importantly, aliquoting is generally unnecessary for -20°C storage, which simplifies laboratory handling protocols. Some preparations (e.g., 20μl sizes) may contain 0.1% BSA for added stability . Always check manufacturer guidelines for specific storage requirements as they may vary between suppliers.

What is the molecular weight of POGZ protein, and how does this affect antibody detection?

POGZ has a calculated molecular weight of approximately 155 kDa, which matches the observed molecular weight in experimental conditions . This consistency between calculated and observed weights suggests minimal post-translational modifications affecting size. When performing Western blot experiments, researchers should optimize gel concentration and running conditions for proteins of this size. Typically, 6-8% SDS-PAGE gels provide optimal resolution for proteins in this molecular weight range. Transfer conditions should also be adjusted accordingly, often requiring longer transfer times or specialized buffers for efficient transfer of larger proteins.

What are the optimal sample preparation protocols for different POGZ antibody applications?

Different experimental approaches require specific optimization strategies:

For Western Blotting:

• Use RIPA or NP-40 lysis buffers containing protease inhibitors

• Load 20-30 μg of total protein per lane

• Include phosphatase inhibitors if phosphorylation status is relevant

• For brain tissues, use specialized neural tissue lysis buffers with gentle homogenization

For Immunohistochemistry:

• For paraffin-embedded tissues, antigen retrieval with TE buffer pH 9.0 is recommended

• Alternatively, citrate buffer pH 6.0 may be used for antigen retrieval

• Mouse brain and testis tissues have shown positive staining results

• Human prostate cancer and kidney tissues have also been successfully stained

For Immunofluorescence/ICC:

• Fixation with 4% paraformaldehyde for 15-20 minutes

• Permeabilization with 0.1-0.3% Triton X-100

• HepG2 and U-251 cell lines have shown robust staining results

Appropriate controls should always be included to validate specificity.

How should researchers address potential cross-reactivity in POGZ antibody experiments?

Cross-reactivity remains a critical consideration in antibody-based experiments. For POGZ antibodies:

• Pre-absorption tests: Incubate antibody with excess recombinant POGZ protein prior to primary staining to confirm specificity.

• Knockout/knockdown controls: Use POGZ knockout or knockdown samples as negative controls. Research has validated POGZ antibodies using Emx1-Pogz and Gad2-Pogz conditional knockout mice where POGZ protein was barely detected in cortical neurons of Emx1-Pogz mice compared to wild-type .

• Species specificity: Validated POGZ antibodies show reactivity with human and mouse samples . When working with other species, preliminary validation is essential.

• Isotype controls: Include matched isotype controls (e.g., Rabbit IgG for polyclonal rabbit antibodies) to identify non-specific binding.

• Tissue-specific optimization: Different tissues may require modified protocols; for example, brain tissue requires careful optimization of fixation and permeabilization protocols.

What are the recommended dilution ranges for POGZ antibodies in different applications?

Optimal dilutions vary by application type and specific antibody preparation. The following table summarizes recommended ranges based on validated research:

It's critical to note that these ranges serve as starting points, and each experimental system requires individual optimization . Sample-dependent variations may necessitate adjustments. Always perform dilution series experiments when setting up a new system.

How can POGZ antibodies be utilized to study neurodevelopmental disorders?

POGZ has been implicated in various neurodevelopmental disorders, making antibody-based studies valuable for understanding disease mechanisms:

• Developmental expression profiling: Research shows POGZ expression is strongest at embryonic day 13 and gradually decreases throughout brain development, suggesting critical roles in early neural development . Researchers can use POGZ antibodies with developmental tissue series to map expression changes across critical neurodevelopmental windows.

• Cell-type specific analyses: POGZ is enriched in cerebrocortical and hippocampal neurons during early developmental stages, with nuclear expression also detected in Purkinje cells in cerebellum at postnatal days 7 and 15, disappearing by day 30 . Immunohistochemistry with neuronal subtype markers can reveal cell-specific expression patterns relevant to developmental disorders.

• Subcellular localization studies: In cultured hippocampal neurons, POGZ is primarily nuclear but also present in axons and dendrites with partial synaptic localization . Co-localization studies with synaptic markers can help elucidate roles in synaptic function.

• Knockout model validation: Conditional knockout mice (Emx1-Pogz and Gad2-Pogz) provide valuable tools for studying POGZ function in specific neuronal populations . POGZ antibodies are essential for validating knockout efficiency and studying compensatory mechanisms.

• Patient-derived samples: POGZ mutations are associated with Mental Retardation, Autosomal Dominant 37 . Antibodies can be used to assess expression and localization differences in patient-derived cells or tissues.

What techniques can be employed to study POGZ interactions with other proteins?

Understanding POGZ interactions is crucial for elucidating its functional roles:

• Co-immunoprecipitation (Co-IP): POGZ antibodies have been validated for IP applications in HeLa cells . Researchers can use these for Co-IP studies to identify interacting partners. POGZ has been shown to interact with CBX5 (HP1α) to regulate aurora kinase B activation and chromosome segregation .

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity. Combining POGZ antibodies with antibodies against suspected interactors enables spatial mapping of interactions within cellular compartments.

• Chromatin immunoprecipitation (ChIP): Since POGZ functions in chromatin remodeling, ChIP assays using POGZ antibodies can identify genomic binding sites and co-occupied regions with other transcription factors.

• FRET/BRET analysis: For studying dynamic interactions, fluorescence or bioluminescence resonance energy transfer techniques can be employed using tagged proteins and validated with antibody detection.

• Mass spectrometry following IP: Immunoprecipitation with POGZ antibodies followed by mass spectrometry analysis can identify novel interacting partners in an unbiased manner.

• Yeast two-hybrid validation: POGZ interaction with transcription factor SP1 was identified in a yeast two-hybrid system . Antibodies can validate such interactions in mammalian contexts.

How can contradictory results in POGZ expression studies be reconciled?

When researchers encounter conflicting POGZ expression data, several methodological approaches can help resolve discrepancies:

• Antibody epitope analysis: Different antibodies target distinct regions of POGZ. The 30106-1-AP antibody uses a POGZ fusion protein immunogen , while PACO29046 targets the recombinant human POGZ (1-300AA) region . Epitope differences may explain discrepant results, especially if alternative splicing or post-translational modifications affect antibody recognition.

• Isoform-specific detection: POGZ has multiple alternatively spliced isoforms . Using PCR primers or antibodies that distinguish between isoforms can clarify whether apparently contradictory results actually reflect different isoform detection.

• Developmental timing: POGZ expression changes dramatically during development. Expression is strong at embryonic day 13 but decreases throughout brain development . Results from different developmental stages should not be directly compared without acknowledging temporal regulation.

• Cell-type heterogeneity: POGZ expression varies between neuronal subtypes. For instance, in Emx1-Pogz conditional knockout mice, POGZ was barely detectable in cortical neurons but strongly expressed in medium spiny neurons in the striatum . Tissue heterogeneity must be considered when interpreting expression data.

• Experimental technique comparison: Different techniques (WB, IHC, IF) have varying sensitivity and specificity. Quantitative comparisons should ideally be made using the same technique with appropriate controls.

What are common troubleshooting strategies for weak or absent POGZ antibody signals?

When experiencing weak or absent signals with POGZ antibodies, consider these methodological solutions:

• Antibody concentration optimization:

  • For Western blot, test a dilution series (e.g., 1:1000, 1:2000, 1:4000)

  • For IHC, begin with higher concentrations (1:50) and titrate as needed

• Antigen retrieval enhancement:

  • For FFPE tissues, use TE buffer at pH 9.0 as recommended for POGZ antibodies

  • Extend retrieval time or test alternative methods (e.g., citrate buffer pH 6.0)

• Sample preparation refinement:

  • Ensure fresh samples and proper lysis with protease inhibitors

  • For brain tissues, minimize freeze-thaw cycles and optimize homogenization methods

• Detection system enhancement:

  • Switch to more sensitive detection methods (e.g., from chromogenic to fluorescent)

  • Use signal amplification systems like tyramide signal amplification

  • Extend primary antibody incubation time (overnight at 4°C)

• Fixation optimization:

  • Test different fixation protocols, as overfixation can mask epitopes

  • For IF/ICC applications, compare 4% PFA vs. methanol fixation

• Blocking optimization:

  • Test different blocking reagents (BSA, normal serum, commercial blockers)

  • Extend blocking time to reduce background that might obscure specific signals

How can researchers validate POGZ antibody specificity for their experimental system?

Rigorous validation ensures reliable research outcomes:

• Genetic models: Utilize knockout or knockdown systems as definitive negative controls. Conditional knockout mice (Emx1-Pogz and Gad2-Pogz) show specific deletion patterns that can validate antibody specificity .

• Peptide competition: Pre-incubate antibody with the immunizing peptide to confirm signal reduction or elimination.

• Multiple antibodies: Use antibodies targeting different POGZ epitopes to confirm consistent localization and expression patterns.

• Overexpression systems: Compare endogenous signal with overexpressed POGZ (tagged or untagged) to confirm signal enhancement.

• RNA-protein correlation: Compare protein detection with mRNA expression (qPCR, RNA-seq) to confirm concordance.

• Cell line panel: Test antibody performance across multiple cell lines with known POGZ expression levels (e.g., HeLa, HepG2, SH-SY5Y cells have shown positive Western blot results ).

• Molecular weight verification: Confirm that detected bands match the expected 155 kDa size of POGZ .

What methodological considerations are important when using POGZ antibodies in brain tissue analysis?

Brain tissue presents unique challenges for antibody-based studies:

• Developmental timing: POGZ expression varies significantly during development, with strong expression at embryonic day 13 that gradually decreases throughout development . Age-matched controls are essential for comparative studies.

• Regional specificity: POGZ is enriched in cerebrocortical and hippocampal neurons during early development, with specific patterns in Purkinje cells in the cerebellum at P7 and P15 but disappearing by P30 . Region-specific optimization may be necessary.

• Antigen retrieval optimization: For brain tissue immunohistochemistry, TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 provides an alternative .

• Perfusion fixation: For animal studies, transcardial perfusion with fixative prior to tissue collection improves antibody penetration and signal quality.

• Section thickness optimization: For IHC/IF in brain tissue, section thickness affects antibody penetration. Typically, 20-40 μm sections for free-floating IHC and 5-10 μm sections for slide-mounted applications provide optimal results.

• Autofluorescence reduction: Brain tissue often exhibits high autofluorescence, especially in older animals. Treatments with Sudan Black B or commercial autofluorescence reducers may improve signal-to-noise ratio in fluorescent applications.

• Co-staining with cell-type markers: Combine POGZ antibody with markers for neurons (NeuN), astrocytes (GFAP), or other neural cell types to clarify cell-specific expression patterns.

How might POGZ antibodies be utilized in studying neurodevelopmental disorder mechanisms?

POGZ mutations are associated with neurodevelopmental disorders including Mental Retardation, Autosomal Dominant 37 (MRD37) . Future research directions include:

• Patient-derived models: Using POGZ antibodies to analyze expression, localization, and interaction differences in patient-derived iPSCs, neural progenitors, and differentiated neurons.

• Synaptic function studies: Since POGZ localizes to synapses in hippocampal neurons , antibodies can be used to investigate its role in synaptic development, plasticity, and dysfunction in disease models.

• Circuit-specific analyses: Combining POGZ immunostaining with circuit tracing techniques to understand circuit-specific vulnerability in neurodevelopmental disorders.

• Developmental trajectory mapping: Using POGZ antibodies to map altered developmental expression trajectories in disease models to identify critical windows for potential intervention.

• Chromatin regulation dynamics: Since POGZ functions in chromatin remodeling, ChIP-seq studies using POGZ antibodies can reveal altered genomic binding in disease states.

• Therapeutic target validation: POGZ antibodies can help validate and monitor the effects of potential therapeutic approaches targeting POGZ or its downstream pathways.

What advanced techniques can incorporate POGZ antibodies for high-resolution analyses?

Emerging technologies offer new possibilities for POGZ research:

• Super-resolution microscopy: Techniques like STORM, PALM, or STED microscopy combined with POGZ antibodies can reveal nanoscale distribution within nuclear domains or synaptic structures.

• Expansion microscopy: Physical expansion of specimens combined with POGZ immunolabeling can provide enhanced spatial resolution of protein localization.

• APEX2 proximity labeling: Fusion of APEX2 to POGZ followed by biotinylation and antibody-based detection can map proximal proteins in different cellular compartments.

• CUT&RUN/CUT&Tag: These techniques provide higher resolution than conventional ChIP for mapping chromatin interactions using POGZ antibodies.

• Single-cell proteomics: POGZ antibodies can be incorporated into emerging single-cell proteomic workflows to understand cell-to-cell variability in expression and modifications.

• Live-cell imaging: Combining traditional fixed-cell antibody validation with live-cell approaches using fluorescent protein fusions can provide dynamic insights into POGZ behavior.

• Spatial transcriptomics correlation: Correlating POGZ antibody staining patterns with spatial transcriptomics data can reveal regional relationships between POGZ protein and regulated genes.

How can quantitative analyses of POGZ expression be standardized across research studies?

Standardizing POGZ quantification will enhance research reproducibility:

• Reference standard inclusion: Include recombinant POGZ protein standards of known concentration in Western blots for absolute quantification.

• Normalization strategy consensus: Establish consistent loading controls for different applications (e.g., GAPDH/β-actin for WB, DAPI/total protein stains for IHC/IF).

• Digital image analysis protocols: Develop standardized image acquisition and analysis protocols for IF/IHC quantification, including threshold setting and background subtraction methods.

• Multi-antibody validation: Confirm quantitative findings with multiple antibodies targeting different POGZ epitopes.

• Absolute quantification methods: Implement absolute quantification techniques such as selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) mass spectrometry with antibody-based enrichment.

• Technical replicate guidelines: Establish minimum requirements for technical and biological replicates in quantitative POGZ studies.

• Developmental stage reporting: Given the developmental regulation of POGZ, standardize age/developmental stage reporting for neurodevelopmental studies.