FZR1 Antibody

What is FZR1 and what are its primary cellular functions?

FZR1 (Fizzy-related protein homolog, also known as CDH1, FYR, KIAA1242, or CDC20-like protein 1) functions as a substrate-specific adapter for the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin-protein ligase complex . It associates with the APC/C in late mitosis, replacing CDC20, and activates the APC/C during anaphase and telophase. This activation ensures that positive cell cycle regulators do not accumulate prematurely .

FZR1 undergoes phosphorylation at the G1/S transition, leading to its dissociation from the APC/C. Following DNA damage, it contributes to the G2 DNA damage checkpoint through dephosphorylation and reassociation with APC/C, resulting in PLK1 ubiquitination that prevents mitotic entry . FZR1 also targets the DNA-end resection factor RBBP8/CtIP for ubiquitination and proteasomal degradation, suggesting a role in favoring error-prone non-homologous end joining over error-free homologous recombination during DNA repair .

What experimental applications are FZR1 antibodies typically used for?

FZR1 antibodies are commonly used in multiple research applications:

• Western Blotting (WB): For detecting FZR1 protein expression levels in various tissues and cell lines

• Immunohistochemistry on paraffin-embedded sections (IHC-P): For visualizing FZR1 localization in tissue samples

• Immunofluorescence (ICC/IF): For examining subcellular localization, showing nuclear expression in cells like HeLa

• ELISA: For quantitative detection of FZR1

• Proximity Ligation Assay (PLA): For studying protein-protein interactions involving FZR1

These applications enable researchers to investigate FZR1 expression, localization, and interactions in both physiological and pathological contexts.

What species reactivity is available for commercial FZR1 antibodies?

Commercial FZR1 antibodies display varying species reactivity profiles:

When selecting an FZR1 antibody, researchers should verify the specific reactivity needed for their experimental model system. Cross-reactivity information is typically determined through sequence homology analysis and experimental validation .

How can FZR1 antibodies be used to investigate neurodevelopmental disorders?

FZR1 has emerged as a significant factor in neurodevelopment and neurological disorders. For researchers investigating these connections, FZR1 antibodies provide critical tools for:

• Analyzing FZR1 expression patterns in neural tissues and developmental time points

• Investigating protein-protein interactions in neuronal cells through co-immunoprecipitation or proximity ligation assays

• Examining post-translational modifications that affect FZR1 function in neural contexts

Recent research has identified de novo missense variants in FZR1 associated with developmental and epileptic encephalopathies (DEE), including childhood-onset generalized epilepsy and intellectual disability . Using Drosophila models with mutant alleles of the FZR1 homologue (fzr), researchers demonstrated that patient variants affected proper neurodevelopment, supporting a loss-of-function mechanism .

For such studies, researchers should consider using antibodies that can detect specific phosphorylation states of FZR1, as its phosphorylation status regulates its association with the APC/C complex and subsequent functions in both dividing and post-mitotic neurons.

What methodological considerations are important when using FZR1 antibodies in cancer research?

When investigating FZR1 in cancer contexts, particularly as a potential biomarker for breast cancer neoadjuvant chemotherapy response , researchers should consider:

• Sample preparation optimization:

  • For paraffin-embedded tissues, antigen retrieval methods may significantly affect FZR1 detection

  • Standardization of fixation protocols is critical for comparative studies

• Controls for specificity:

  • Include FZR1 knockdown/knockout samples as negative controls

  • Compare nuclear versus cytoplasmic fractions, as FZR1 shows nuclear localization

• Quantification approaches:

  • Implement digital image analysis for IHC quantification rather than subjective scoring

  • Consider multiplexed immunofluorescence to correlate FZR1 expression with other cancer markers

• Clinical correlation methods:

  • Design studies that correlate FZR1 expression levels with treatment response data

  • Account for tumor heterogeneity by analyzing multiple regions of each sample

Recent research suggests FZR1 may serve as a novel biomarker for predicting response to neoadjuvant chemotherapy in breast cancer, addressing a critical need in personalized treatment approaches .

What are the best protocols for detecting protein-protein interactions involving FZR1?

Investigating FZR1's interactions with APC/C components and substrates requires specialized approaches:

• Proximity Ligation Assay (PLA):

  • Optimal for detecting endogenous protein interactions in situ

  • Requires antibodies raised in different species against FZR1 and its potential interaction partners

  • Provides visualization of interactions with subcellular resolution

• Co-immunoprecipitation approaches:

  • Use lysis buffers that preserve native protein complexes (avoid harsh detergents)

  • Consider reversible crosslinking to stabilize transient interactions

  • Validate pull-downs with reciprocal immunoprecipitations

• FRET/BRET analysis:

  • For dynamic, real-time interaction studies in living cells

  • Requires fusion protein construction with appropriate tags

When investigating FZR1 interactions with ubiquitination targets such as RBBP8/CtIP , researchers should additionally consider proteasome inhibition to stabilize these often transient interactions.

How should researchers select the optimal FZR1 antibody epitope for specific applications?

The choice of antibody epitope is critical for experimental success with FZR1:

Researchers should align their epitope selection with:

• The specific protein region of interest (e.g., functional domains)

• Post-translational modifications that might mask epitopes

• Accessibility in folded/native vs. denatured states

• Sequence conservation if working across species

For cell cycle studies where FZR1 phosphorylation status is critical, phospho-specific antibodies may be necessary to distinguish between active and inactive forms of the protein .

What are the optimal fixation and permeabilization methods for FZR1 immunofluorescence?

For successful immunofluorescence detection of FZR1:

• Fixation protocol:

  • Paraformaldehyde (4%) at room temperature for 15 minutes is effective for preserving FZR1 localization in HeLa cells

  • Methanol fixation may improve nuclear antigen accessibility but test empirically

• Permeabilization considerations:

  • Mild detergents (0.1-0.2% Triton X-100) are generally sufficient

  • Brief (5-10 minute) permeabilization to avoid epitope damage

• Blocking optimization:

  • BSA (3-5%) with normal serum matching secondary antibody host

  • Extended blocking (1-2 hours) may reduce background

• Antibody dilution and incubation:

  • 1:500 dilution has been effective for certain FZR1 antibodies

  • Overnight incubation at 4°C may improve signal-to-noise ratio

For co-localization studies, counterstaining with markers such as phalloidin (cytoskeleton) can provide valuable context for interpreting FZR1 localization patterns .

How can researchers troubleshoot non-specific binding or weak signals when using FZR1 antibodies?

When encountering issues with FZR1 antibody performance:

• For non-specific binding:

  • Increase antibody dilution incrementally (e.g., from 1:500 to 1:1000)

  • Add 0.1-0.2% Tween-20 to washing buffers

  • Include validation controls using FZR1-depleted samples

  • Pre-absorb antibody with recombinant FZR1 protein (for polyclonal antibodies)

• For weak signals:

  • Optimize antigen retrieval for fixed tissues (test citrate vs. EDTA buffers)

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

  • Use signal amplification systems (e.g., tyramide signal amplification)

  • Check sample preparation to ensure target protein is preserved

• For inconsistent results:

  • Standardize cell/tissue harvesting conditions (FZR1 levels fluctuate with cell cycle)

  • Control for phosphorylation status using phosphatase inhibitors in lysates

  • Validate lot-to-lot antibody consistency with standard samples

Remember that FZR1 expression and localization changes during cell cycle progression, so synchronizing cells may be necessary for consistent results in certain experiments.

How should researchers interpret contradictory FZR1 expression patterns across different experimental systems?

When faced with contradictory FZR1 expression data:

• Consider cell cycle variation:

  • FZR1 activity and levels are regulated throughout the cell cycle

  • Unsynchronized populations may show mixed expression patterns

  • Document cell synchronization methods and cell cycle stage verification

• Evaluate antibody specificity:

  • Different antibodies may recognize distinct FZR1 isoforms or modified forms

  • Compare results using antibodies targeting different epitopes

  • Include knockout/knockdown validation controls

• Assess experimental context:

  • FZR1 function differs in neuronal vs. proliferating cells

  • Disease states may alter expression or localization patterns

  • Different tissues show varying baseline expression levels

• Technical variables:

  • Compare protocols for sample preparation, especially fixation methods

  • Standardize quantification approaches across experiments

  • Document exposure settings for immunofluorescence or blot development

For integrating contradictory findings across models, consider systems biology approaches that place FZR1 within its functional network context rather than examining it in isolation.

What methods provide the most rigorous validation of FZR1 antibody specificity?

To ensure antibody specificity for FZR1:

• Genetic approaches:

  • CRISPR/Cas9 knockout cell lines as negative controls

  • siRNA knockdown with demonstration of signal reduction

  • Rescue experiments with exogenous FZR1 expression

• Biochemical validation:

  • Immunoprecipitation followed by mass spectrometry

  • Peptide competition assays using the immunizing peptide

  • Multiple antibodies targeting different epitopes should show consistent patterns

• Expression system controls:

  • Overexpression systems with tagged FZR1 constructs

  • Inducible expression systems to demonstrate signal correlation with expression

  • Heterologous expression in null backgrounds

• Cross-reactivity assessment:

  • Testing in tissues from multiple species with predicted reactivity

  • Evaluating signal in tissues known to lack FZR1 expression

  • Testing related family members to ensure specificity

How can researchers differentiate between FZR1 isoforms or post-translationally modified forms?

To distinguish between different forms of FZR1:

• Isoform differentiation:

  • Use antibodies targeting isoform-specific sequences

  • Combine with RT-PCR to correlate protein detection with transcript expression

  • Consider 2D gel electrophoresis to separate isoforms by both MW and pI

• Phosphorylation analysis:

  • Phospho-specific antibodies for key regulatory sites

  • Phosphatase treatment of samples as controls

  • Lambda phosphatase treatment to confirm phosphorylation-dependent mobility shifts

• Ubiquitination detection:

  • Immunoprecipitation under denaturing conditions

  • Probing for ubiquitin after FZR1 immunoprecipitation

  • Proteasome inhibitor treatment to stabilize ubiquitinated forms

• Advanced techniques:

  • Mass spectrometry for comprehensive PTM mapping

  • Phos-tag gels to separate phosphorylated from non-phosphorylated forms

  • Targeted mass spectrometry using multiple reaction monitoring

Since FZR1 phosphorylation status significantly affects its activity in the APC/C complex , researchers studying cell cycle regulation should particularly focus on methods that can distinguish active from inactive FZR1 forms.

How are FZR1 antibodies being used to explore its role in neurodevelopmental disorders?

Recent discoveries have linked FZR1 to neurodevelopmental disorders:

• Clinical connections:

  • De novo missense variants in FZR1 have been identified in patients with developmental and epileptic encephalopathies (DEE)

  • These patients exhibit childhood-onset generalized epilepsy, intellectual disability, and mild ataxia

• Functional studies using antibodies:

  • Immunohistochemistry to map FZR1 expression in developing neural tissues

  • Co-localization with neuronal markers to identify affected cell populations

  • Protein interaction studies to identify neural-specific binding partners

• Mechanistic investigations:

  • Examining how patient variants affect protein stability and localization

  • Assessing changes in substrate targeting using co-immunoprecipitation

  • Monitoring post-mitotic functions in neurons specifically

These approaches have helped establish FZR1's role beyond cell cycle regulation, revealing important functions in neurodevelopment that can be disrupted in pathological conditions .

What methodological advances are improving FZR1 antibody-based cancer biomarker research?

FZR1's emerging role as a biomarker in cancer research, particularly for breast cancer neoadjuvant chemotherapy response , has driven methodological innovations:

• Digital pathology integration:

  • Automated scoring of FZR1 immunohistochemistry

  • Machine learning algorithms to correlate expression patterns with outcomes

  • Standardized quantification across multi-center trials

• Multiplexed detection approaches:

  • Sequential immunofluorescence to examine FZR1 alongside other markers

  • Mass cytometry (CyTOF) for single-cell protein profiling

  • Spatial transcriptomics combined with protein detection

• Liquid biopsy development:

  • Detection of FZR1 in circulating tumor cells

  • Correlation with tissue expression patterns

  • Longitudinal monitoring during treatment

• Functional predictive assays:

  • Ex vivo drug response testing correlated with FZR1 expression

  • Patient-derived organoids to validate FZR1 as a response predictor

  • Computational models incorporating FZR1 expression data

These approaches aim to translate FZR1's biological role into clinically actionable information for personalized treatment decisions, particularly in determining which patients will benefit from neoadjuvant chemotherapy .

How can researchers design experiments to distinguish between cell cycle-dependent and independent functions of FZR1?

To delineate FZR1's diverse functions:

• Cell synchronization approaches:

  • Compare FZR1 interactions in synchronized vs. asynchronous populations

  • Use thymidine block, serum starvation, or cell sorting to isolate specific cell cycle phases

  • Combine with cell cycle markers (cyclin B, phospho-histone H3) in co-staining experiments

• Post-mitotic cell studies:

  • Examine FZR1 in terminally differentiated neurons or other non-dividing cells

  • Compare substrate targeting between proliferating and non-proliferating cells

  • Use inducible degradation systems to remove FZR1 at specific cell cycle stages

• Domain-specific mutations:

  • Generate separation-of-function mutations that affect specific FZR1 activities

  • Use antibodies to track the localization and interactions of these mutants

  • Correlate with functional outcomes in different cellular contexts

• Computational analysis:

  • Network analysis to identify context-specific FZR1 interaction partners

  • Correlation of expression patterns with cell-cycle dependent and independent processes

  • Pathway enrichment to distinguish between different functional roles

This research direction is particularly important given FZR1's emerging roles beyond classic cell cycle regulation, including neurodevelopment and cancer progression .