ZNF207 Antibody, Biotin conjugated

What is ZNF207 and what are its known biological functions?

ZNF207 (Zinc Finger Protein 207), also known as BuGZ (BUB3-interacting and GLEBS motif-containing protein), is a multifunctional protein involved in several critical cellular processes. It functions primarily as a kinetochore- and microtubule-binding protein that plays a key role in spindle assembly during mitosis . The protein is mainly composed of disordered low-complexity regions and can undergo phase transition or coacervation to form temperature-dependent liquid droplets .

ZNF207's coacervation promotes microtubule bundling and concentrates tubulin, which subsequently enhances microtubule polymerization and assembly of spindle and spindle matrix by concentrating essential building blocks . Additionally, ZNF207 acts as a regulator of mitotic chromosome alignment by mediating the stability and kinetochore loading of BUB3 . Recent research has also identified ZNF207 as an important factor in stem cell biology, where it contributes to the maintenance of human embryonic stem cells (hESCs) and facilitates reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) .

What are the structural features of biotin-conjugated ZNF207 antibodies?

Biotin-conjugated ZNF207 antibodies are immunoglobulins (typically IgG) that have been chemically modified with biotin molecules to enhance detection sensitivity and versatility in various experimental applications . These antibodies are commonly developed by immunizing rabbits with recombinant human BUB3-interacting and GLEBS motif-containing protein ZNF207 (often using amino acids 206-463) .

The typical structural features include:

• A polyclonal IgG antibody core raised against ZNF207 epitopes

• Biotin molecules conjugated to the antibody structure

• Storage in a preservative buffer (commonly containing 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4)

The biotin conjugation offers significant advantages in detection systems due to the extremely high affinity interaction between biotin and streptavidin/avidin, which can be leveraged for signal amplification in multiple experimental contexts .

How does the biotin conjugation affect antibody function and application versatility?

Biotin conjugation significantly enhances the versatility of ZNF207 antibodies by enabling compatibility with various detection systems without compromising epitope recognition capabilities . The biotin-streptavidin interaction is one of the strongest non-covalent interactions in nature (Kd ≈ 10^-15 M), providing several methodological advantages:

• Signal amplification: Multiple streptavidin molecules (conjugated to enzymes, fluorophores, or other reporters) can bind to a single biotinylated antibody, enhancing detection sensitivity

• Compatibility with diverse detection systems: The same biotinylated antibody can be used with various streptavidin-conjugated reporters

• Reduced background: The specificity of biotin-streptavidin interaction helps minimize non-specific binding

What are the optimal applications for biotin-conjugated ZNF207 antibodies?

Biotin-conjugated ZNF207 antibodies have been validated primarily for ELISA applications, but their utility extends to other techniques where biotinylated reagents offer advantages . The optimal applications include:

For applications beyond ELISA, researchers should perform validation experiments to confirm antibody performance, as most commercial sources specifically validate these antibodies for ELISA applications only .

What are the recommended protocols for using biotin-conjugated ZNF207 antibodies in ELISA?

When using biotin-conjugated ZNF207 antibodies in ELISA, the following protocol framework is recommended:

• Plate preparation:

  • Coat high-binding ELISA plates with capture antibody or target antigen

  • Incubate overnight at 4°C

  • Wash 3-5 times with washing buffer (PBS + 0.05% Tween-20)

• Blocking:

  • Block with 3-5% BSA or non-fat milk in PBS for 1-2 hours at room temperature

  • Wash 3-5 times

• Sample incubation:

  • Add samples containing ZNF207 protein

  • Incubate for 1-2 hours at room temperature

  • Wash 3-5 times

• Antibody incubation:

  • Dilute biotin-conjugated ZNF207 antibody in antibody diluent (typically 1:1000, though optimization may be required)

  • Incubate for 1-2 hours at room temperature

  • Wash 3-5 times

• Detection:

  • Add streptavidin-HRP (typically 1:5000 to 1:10000 dilution)

  • Incubate for 30-60 minutes at room temperature

  • Wash 3-5 times

• Substrate addition and measurement:

  • Add appropriate substrate (TMB for HRP)

  • Monitor color development

  • Stop reaction with stop solution (typically 2N H₂SO₄)

  • Read absorbance at appropriate wavelength (450nm for TMB)

Important considerations include using appropriate negative controls (samples without target protein) and ensuring no endogenous biotin is present in samples, which could interfere with detection .

How can researchers optimize Western blot protocols when using anti-ZNF207 antibodies?

While the biotin-conjugated ZNF207 antibodies are primarily validated for ELISA, related anti-ZNF207 antibodies can be used effectively in Western blot applications with the following optimization strategies :

• Sample preparation:

  • Extract proteins using RIPA or NP-40 based lysis buffers

  • Include protease inhibitors to prevent degradation

  • Denature samples at 95°C for 5 minutes in Laemmli buffer

• Gel electrophoresis and transfer:

  • Use 10-12% polyacrylamide gels (ZNF207 has a molecular weight of approximately 51 kDa)

  • Transfer to PVDF membrane at 100V for 60-90 minutes or 25V overnight

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Dilute primary anti-ZNF207 antibody at 1:500-1:1000 in antibody diluent

  • Incubate overnight at 4°C

  • Wash 3-5 times with TBST

• Detection:

  • If using biotin-conjugated antibody: Incubate with streptavidin-HRP (1:5000-1:10000)

  • If using unconjugated antibody: Incubate with HRP-conjugated secondary antibody (1:5000-1:10000)

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expected band size: 51 kDa

Optimization techniques for challenging Western blots include:

• Increasing antibody concentration for weak signals

• Extending incubation times or using higher protein loads

• Implementing signal enhancement systems for low-abundance targets

• Using freshly prepared samples to minimize degradation

How can researchers investigate ZNF207's role in spindle assembly using biotin-conjugated antibodies?

ZNF207 (BuGZ) plays a critical role in spindle assembly during mitosis, making it an important target for cell cycle research . To investigate this function using biotin-conjugated antibodies, researchers can implement the following methodologies:

• Immunofluorescence microscopy:

  • Fix cells using 4% paraformaldehyde

  • Permeabilize with 0.2% Triton X-100

  • Block with 3% BSA

  • Incubate with biotin-conjugated ZNF207 antibody

  • Detect using streptavidin-fluorophore conjugates

  • Co-stain with markers for microtubules (anti-α-tubulin) and DNA (DAPI)

  • Analyze co-localization of ZNF207 with spindle structures during various mitotic phases

• Proximity ligation assays (PLA):

  • Use biotin-conjugated ZNF207 antibody in combination with antibodies against potential interacting partners (such as BUB3)

  • Implement PLA protocol to visualize protein-protein interactions in situ

  • Quantify interaction frequency during different cell cycle stages

• Live-cell imaging:

  • Combine antibody-based detection with live-cell imaging techniques

  • Monitor ZNF207 dynamics during spindle assembly in real-time

  • Correlate ZNF207 localization with spindle formation events

When analyzing results, researchers should focus on ZNF207's distribution pattern during prophase, metaphase, anaphase, and telophase, with particular attention to its colocalization with kinetochores and microtubules .

What experimental approaches can elucidate ZNF207's interaction with BUB3 in chromosome alignment?

ZNF207 regulates mitotic chromosome alignment by mediating the stability and kinetochore loading of BUB3 . To investigate this interaction, researchers can employ several complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Lyse synchronized mitotic cells

  • Perform IP using anti-ZNF207 antibodies

  • Analyze precipitates for BUB3 presence by Western blot

  • Compare results under normal conditions versus after depletion of ZNF207

• Chromosome spread analysis:

  • Implement ZNF207 knockdown using siRNA

  • Prepare chromosome spreads from control and knockdown cells

  • Analyze chromosome alignment defects

  • Quantify BUB3 localization at kinetochores using immunofluorescence

• Domain mapping experiments:

  • Create truncated or mutated ZNF207 constructs

  • Express these constructs in cells depleted of endogenous ZNF207

  • Assess which domains are required for BUB3 interaction and stability

  • Correlate interaction capability with functional chromosome alignment

• Proteasome inhibition studies:

  • Treat cells with proteasome inhibitors (e.g., MG132)

  • Analyze whether BUB3 stability is affected by ZNF207 depletion under these conditions

  • This approach can help distinguish between competing models of how ZNF207 stabilizes BUB3

The results from these experiments should be analyzed in the context of two competing models: one suggesting ZNF207 blocks ubiquitination and proteasomal degradation of BUB3, and another indicating the stabilization is proteasome-independent .

How can researchers investigate the phase transition properties of ZNF207 and their functional significance?

ZNF207 (BuGZ) undergoes phase transition or coacervation to form temperature-dependent liquid droplets, which is critical for its function in spindle assembly . To investigate this unique property, researchers can employ the following experimental approaches:

• In vitro phase separation assays:

  • Purify recombinant ZNF207 protein

  • Observe droplet formation under varying conditions (temperature, salt concentration, pH)

  • Perform fluorescence recovery after photobleaching (FRAP) to assess droplet dynamics

  • Add labeled tubulin to assess recruitment to ZNF207 droplets

• Live-cell imaging of phase transition:

  • Express fluorescently-tagged ZNF207 in cells

  • Observe droplet formation in real-time under various conditions

  • Use temperature-controlled microscopy to study temperature-dependence

  • Correlate droplet formation with spindle assembly

• Structure-function analysis:

  • Create mutants lacking specific domains or with altered low-complexity regions

  • Assess their ability to undergo phase separation

  • Correlate phase separation capacity with functional outcomes in spindle assembly

  • Implement domain-swapping experiments to identify critical regions

• Correlative light and electron microscopy (CLEM):

  • Visualize ZNF207 droplets using fluorescence microscopy

  • Examine the same structures at ultrastructural level using electron microscopy

  • Characterize the internal organization of droplets and their interaction with cellular structures

When analyzing results, researchers should focus on how phase separation correlates with functional outcomes, particularly in microtubule bundling and spindle assembly, and how this property distinguishes ZNF207 from other spindle assembly factors .

How can researchers investigate ZNF207's role in maintaining pluripotency in stem cells?

Recent research has revealed that ZNF207 plays a crucial role in maintaining human embryonic stem cell (hESC) pluripotency and self-renewal . To investigate this function, researchers can implement several experimental approaches:

• Gene expression analysis after ZNF207 manipulation:

  • Perform ZNF207 knockdown using siRNA or shRNA in hESCs

  • Analyze changes in pluripotency markers (OCT4, NANOG, SOX2) via qPCR and Western blot

  • Conduct RNA-Seq to identify global transcriptome changes

  • Compare results with overexpression studies to establish dose-dependent effects

• Chromatin immunoprecipitation (ChIP) analysis:

  • Use anti-ZNF207 antibodies for ChIP experiments

  • Assess ZNF207 binding to regulatory elements of pluripotency genes

  • Perform ChIP-Seq to identify genome-wide binding sites

  • Compare binding profiles with those of core pluripotency factors (OCT4, SOX2)

• Morphological and functional assessments:

  • Monitor changes in colony morphology after ZNF207 manipulation

  • Perform alkaline phosphatase (AP) staining to assess pluripotency status

  • Analyze expression of surface markers like TRA-1-60

  • Assess differentiation potential through embryoid body formation or directed differentiation

Research has shown that ZNF207 knockdown results in dramatic changes associated with loss of pluripotency, including reduced AP staining and altered localization of TRA-1-60 from even distribution on the cell membrane to a focal point inside the cell .

What methodologies can reveal ZNF207's interactions with core pluripotency factors?

ZNF207 appears to interact with core pluripotency factors OCT4 and SOX2, forming a complex that regulates stem cell maintenance . To investigate these interactions, researchers can employ these methodologies:

• Co-immunoprecipitation (Co-IP) experiments:

  • Perform Co-IP using anti-ZNF207 antibodies and probe for OCT4/SOX2

  • Perform reciprocal Co-IP using anti-OCT4/SOX2 antibodies and probe for ZNF207

  • Include appropriate controls to confirm specificity

  • Analyze interaction dynamics under various conditions (differentiation, stress)

• Motif analysis and DNA binding studies:

  • Analyze ChIP-Seq data to identify enriched motifs in ZNF207 binding sites

  • Compare with known OCT4/SOX2 binding motifs

  • Perform electrophoretic mobility shift assays (EMSA) to confirm binding specificity

  • Use sequential ChIP to identify genomic regions bound by both ZNF207 and OCT4/SOX2

• Protein domain mapping:

  • Create truncated versions of ZNF207 to identify domains required for OCT4/SOX2 interaction

  • Express these constructs in cells and assess interaction capability

  • Correlate interaction capability with functional outcomes in pluripotency maintenance

Research has revealed that the OCT4/SOX2 consensus motif is enriched as the most significant motif in ZNF207 binding sites, with an E-value of 5.3E-059, and there is substantial overlap of OCT4, SOX2, and ZNF207 binding events across the genome of hESCs .

How can researchers assess ZNF207's role in cellular reprogramming to pluripotency?

ZNF207 has been implicated in enhancing the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs) . To investigate this function, researchers can implement these experimental approaches:

• Reprogramming efficiency assays:

  • Implement standard reprogramming protocols (e.g., OSKM factors)

  • Manipulate ZNF207 levels (knockdown or overexpression)

  • Quantify reprogramming efficiency through colony counting and pluripotency marker analysis

  • Compare wild-type ZNF207 versus DNA-binding domain mutants

• Time-course analysis during reprogramming:

  • Monitor ZNF207 expression levels throughout the reprogramming process

  • Analyze correlation with activation of endogenous pluripotency genes

  • Perform ChIP at different time points to track ZNF207 binding to key regulatory elements

  • Identify the temporal sequence of ZNF207 recruitment to pluripotency-associated loci

• Mechanistic studies:

  • Create ZNF207 mutants lacking specific functional domains

  • Assess their ability to enhance reprogramming

  • Correlate functional domains with specific aspects of the reprogramming process

  • Implement domain-swapping experiments to identify critical regions

Research has shown that knockdown of ZNF207 during reprogramming reduces efficiency by approximately tenfold, while overexpression enhances efficiency by about threefold . Importantly, a mutated form of ZNF207 lacking its DNA binding domain fails to promote increased reprogramming efficiency, suggesting that ZNF207 enhances reprogramming through transcriptional regulation .

How can researchers validate antibody specificity for ZNF207 studies?

Ensuring antibody specificity is crucial for obtaining reliable results in ZNF207 research. Researchers can implement these validation strategies:

• Genetic validation approaches:

  • Perform antibody detection in ZNF207 knockdown/knockout samples

  • Compare signal intensity with control samples

  • Verify signal reduction corresponds to mRNA reduction level

  • Include isotype controls to assess non-specific binding

• Epitope competition assays:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare results with non-competed antibody

  • Verify signal reduction in the presence of competing peptide

  • Test across multiple applications (ELISA, WB, IF)

• Cross-reactivity assessment:

  • Test antibody against related zinc finger proteins

  • Analyze potential cross-reactivity with BUB3 or other interaction partners

  • Perform mass spectrometry on immunoprecipitated samples to confirm target identity

  • Test across multiple species if cross-species reactivity is claimed

• Comparison of multiple antibodies:

  • Use antibodies recognizing different epitopes of ZNF207

  • Compare localization patterns and expression levels

  • Concordant results from multiple antibodies strengthen specificity confidence

  • Discordant results warrant further investigation

These validation steps are particularly important for ZNF207 studies as zinc finger proteins often share structural similarities that may lead to cross-reactivity issues .

What strategies can address common technical challenges in ZNF207 experimental systems?

Researchers investigating ZNF207 may encounter several technical challenges. Here are strategies to address common issues:

• Low signal intensity in detection systems:

  • For biotin-conjugated antibodies: Ensure streptavidin detection system is working properly

  • Increase antibody concentration or incubation time

  • Implement signal amplification systems (e.g., TSA)

  • Reduce washing stringency while maintaining specificity

  • Use fresh antibody preparations (avoid repeated freeze-thaw cycles)

• Non-specific background signals:

  • Optimize blocking conditions (test different blocking agents)

  • Include additional washing steps

  • Pre-absorb antibodies with lysates from relevant cells

  • Include appropriate negative controls in all experiments

  • Validate signals with genetic approaches (siRNA knockdown)

• Inconsistent immunoprecipitation results:

  • Optimize cell lysis conditions to maintain protein-protein interactions

  • Include protease and phosphatase inhibitors

  • Test different detergent concentrations

  • Cross-link protein complexes if interactions are transient

  • Optimize salt concentration to maintain specific interactions

• Challenges in detecting phase-separated ZNF207:

  • Carefully control temperature during sample preparation

  • Use fixation methods that preserve membraneless organelles

  • Implement specialized imaging techniques optimized for liquid droplets

  • Consider live-cell imaging to avoid fixation artifacts

Each of these challenges requires systematic troubleshooting and optimization for the specific experimental system being used .

How can researchers integrate multi-omics approaches to comprehensively study ZNF207 functions?

To gain comprehensive insights into ZNF207 functions, researchers can integrate various omics approaches:

• Integrated genomics and proteomics:

  • Combine ChIP-Seq data (ZNF207 binding sites) with RNA-Seq (transcriptional effects)

  • Correlate binding patterns with expression changes after ZNF207 manipulation

  • Perform proteomics on ZNF207 interactome (IP-MS)

  • Integrate data to build comprehensive regulatory networks

• Spatial and temporal profiling:

  • Analyze ZNF207 dynamics across cell cycle phases

  • Combine with single-cell RNA-Seq to assess cell-to-cell variability

  • Implement spatial transcriptomics to examine local effects of ZNF207

  • Correlate with chromatin accessibility data (ATAC-Seq)

• Functional genomics integration:

  • Perform CRISPR screens to identify synthetic interactions with ZNF207

  • Correlate with transcriptome changes and binding patterns

  • Implement network analysis to identify key nodes and pathways

  • Validate predictions with targeted functional assays

• Data analysis and integration pipelines:

  • Implement computational pipelines specifically designed for integration

  • Use machine learning approaches to identify patterns across datasets

  • Develop visualization tools to represent complex multi-dimensional data

  • Apply systems biology approaches to model ZNF207 functions

This integrated approach can provide insights into how ZNF207 functions in different cellular contexts, particularly in its dual roles in mitotic spindle assembly and pluripotency maintenance .

What are the emerging research questions about ZNF207's multifunctional roles?

Research on ZNF207 has revealed its roles in seemingly distinct cellular processes, raising several important questions for future investigation:

• Mechanistic connection between mitotic and pluripotency functions:

  • How does ZNF207 balance its roles in cell division and transcriptional regulation?

  • Are these functions carried out by distinct protein pools or isoforms?

  • How is ZNF207 partitioned between nuclear transcriptional complexes and spindle structures?

  • Do post-translational modifications regulate functional switching?

• Phase separation properties in diverse cellular contexts:

  • How does phase separation contribute to transcriptional regulation versus spindle assembly?

  • Are the biophysical properties of ZNF207 droplets context-dependent?

  • What cellular factors regulate ZNF207 phase transition in different settings?

  • How do disease states affect ZNF207 phase separation properties?

• Evolutionary conservation and specialization:

  • How conserved are ZNF207's dual functions across species?

  • Have different organisms specialized ZNF207 functions differently?

  • What structural features have been conserved versus diverged?

  • How did the pluripotency regulatory function evolve in mammals?

These questions highlight the need for continued research into this multifunctional protein and its diverse cellular roles .

How can advanced imaging technologies enhance our understanding of ZNF207 dynamics?

Emerging advanced imaging technologies offer exciting opportunities to study ZNF207 with unprecedented resolution and detail:

• Super-resolution microscopy approaches:

  • Implement STORM, PALM, or STED microscopy to visualize ZNF207 distribution at nanoscale resolution

  • Examine the internal structure of phase-separated droplets

  • Analyze co-localization with interaction partners with precision beyond the diffraction limit

  • Track the dynamics of single ZNF207 molecules during mitosis or pluripotency maintenance

• Live-cell imaging with optogenetic control:

  • Develop optogenetic tools to manipulate ZNF207 function with spatial and temporal precision

  • Observe immediate consequences of ZNF207 inactivation during specific cellular processes

  • Create photoactivatable ZNF207 variants to study local function in specific cellular compartments

  • Implement FRET-based sensors to monitor ZNF207 interactions in real-time

• Correlative microscopy approaches:

  • Combine fluorescence imaging of ZNF207 with electron microscopy

  • Examine ultrastructural features of ZNF207-containing structures

  • Implement cryo-electron tomography to visualize ZNF207 in its native cellular environment

  • Analyze the relationship between ZNF207 droplets and cellular organelles

These advanced imaging approaches can provide crucial insights into the dynamic behavior of ZNF207 in various cellular contexts, particularly regarding its phase separation properties and interactions with chromatin and spindle structures .

What potential therapeutic applications might emerge from ZNF207 research?

While current research on ZNF207 is primarily fundamental in nature, several potential therapeutic applications might emerge as our understanding deepens:

• Cancer therapeutics targeting mitotic functions:

  • Develop inhibitors targeting ZNF207-BUB3 interaction for cancer therapy

  • Exploit cancer cell dependency on proper chromosome segregation

  • Design molecules that disrupt phase separation properties specifically in cancer cells

  • Create synthetic lethality approaches based on ZNF207 dependencies in specific cancer types

• Stem cell applications based on pluripotency functions:

  • Enhance reprogramming efficiency for therapeutic iPSC generation

  • Improve maintenance of stemness in ex vivo culture systems

  • Develop more efficient differentiation protocols by manipulating ZNF207

  • Create engineered stem cell therapies with controlled ZNF207 expression

• Neurodegenerative disease applications:

  • Investigate potential roles of ZNF207 phase separation in protein aggregation diseases

  • Explore connections between aberrant phase separation and neurodegeneration

  • Develop modulators of ZNF207 phase properties as potential therapeutics

  • Target cell division defects in neural progenitors caused by ZNF207 dysfunction

• Diagnostic applications:

  • Develop ZNF207-based biomarkers for specific cancer types

  • Create diagnostic tools based on ZNF207 expression patterns

  • Implement prognostic indicators based on ZNF207 mutations or expression levels

  • Design companion diagnostics for therapies targeting ZNF207-dependent pathways