enpl-1 Antibody

What is ENPL-1 and why is it significant for research?

ENPL-1 is the Caenorhabditis elegans homolog of GRP94 (HSP90B1), an endoplasmic reticulum (ER) chaperone protein that plays a critical role in insulin processing and secretion. Its significance stems from its involvement in insulin signaling pathways essential for proper development and growth control in C. elegans. Research has demonstrated that ENPL-1 binds to proinsulin/pro-DAF-28 via its client binding domain, facilitating insulin maturation and secretion. Defects in ENPL-1 function have been associated with elevated ER stress, impaired insulin secretion, and phenotypes such as sterility and body size abnormalities, making it a valuable model for studying mechanisms relevant to diabetes and protein processing disorders .

How is ENPL-1 protein expression distributed in C. elegans?

ENPL-1 shows a broad expression pattern throughout C. elegans, with particularly strong expression in neurons, vulva, germline, and intestine. Using CRISPR/CAS9 knock-in of super folder GFP (sfGFP) at the ENPL-1 locus and 3xFlag::ENPL-1 transgene insertion, researchers have established that ENPL-1 is present in a perinuclear pattern characteristic of the ER. The protein is detectable from early developmental stages in the embryo. While ENPL-1 is primarily localized to the ER lumen, subcellular fractionation studies have revealed that a small fraction of the protein can be found outside the ER, suggesting potential non-ER functions that warrant further investigation .

What are the primary applications of ENPL-1 antibodies in C. elegans research?

ENPL-1 antibodies serve multiple experimental purposes in C. elegans research:

• Protein expression analysis: Validating the presence and levels of ENPL-1 in wild-type and mutant strains using Western blot techniques

• Immunoprecipitation studies: Investigating ENPL-1 interactions with binding partners such as proinsulin/pro-DAF-28

• Localization studies: Confirming subcellular distribution when used alongside fluorescent protein tags

• Phenotypic validation: Confirming the specificity of ENPL-1 mutant phenotypes through antibody-based rescue experiments

• Biochemical pathway analysis: Probing insulin secretion pathways and ER stress responses

These applications have contributed to understanding ENPL-1's fundamental role in insulin processing and secretion mechanisms, with broader implications for protein folding disorders and secretory pathway research .

How can one design experiments to investigate ENPL-1's client binding specificity?

Designing experiments to investigate ENPL-1's client binding specificity requires a multifaceted approach:

• Domain mapping experiments: Generate constructs with mutations or deletions in the client binding domain of ENPL-1 to identify critical residues for proinsulin/pro-DAF-28 binding. Research has demonstrated that specific domains of ENPL-1 are required for its interaction with proinsulin, suggesting a targeted binding mechanism rather than general chaperone activity .

• Co-immunoprecipitation assays: Perform co-IP experiments using tagged versions of ENPL-1 (such as 3xFlag::ENPL-1) and potential client proteins, followed by Western blotting or mass spectrometry to identify interacting partners.

• Yeast two-hybrid or mammalian two-hybrid screening: Systematically test interactions between ENPL-1 and candidate proteins to identify novel binding partners.

• In vitro binding assays: Express and purify recombinant ENPL-1 and test direct binding to potential client proteins using techniques such as surface plasmon resonance or isothermal titration calorimetry.

• Competition assays: Test whether known ENPL-1 clients compete for binding, which would suggest shared binding sites or mechanisms.

The experimental approach should include appropriate controls, such as testing binding to other ER chaperones, to establish specificity of the interactions .

What methodological approaches can resolve contradictions between ENPL-1 localization data from different experimental techniques?

Resolving contradictions in ENPL-1 localization data requires employing multiple complementary techniques and careful experimental design:

• Validation with multiple tagging strategies: Studies have shown that ENPL-1 is primarily localized to the ER lumen, but a fraction may exist outside the ER. To resolve potential artifacts from tagging approaches, researchers should compare:

  • N-terminal vs. C-terminal tags

  • Different tag sizes (small epitope tags vs. fluorescent proteins)

  • Knock-in vs. transgenic approaches

• Subcellular fractionation with biochemical verification: Perform careful subcellular fractionation with verification using established compartment markers for ER, cytosol, mitochondria, and other organelles.

• Super-resolution microscopy: Employ techniques like STED or STORM microscopy for higher-resolution localization beyond conventional confocal microscopy.

• Immuno-electron microscopy: Use gold-labeled antibodies against ENPL-1 to visualize localization at ultrastructural resolution.

• Functional validation experiments: Design assays that can detect ENPL-1 activity in different cellular compartments to confirm the biological relevance of localization data.

• Controls for fixation and permeabilization artifacts: Use multiple fixation protocols to rule out redistribution during sample preparation.

Research has indicated that while ENPL-1 is predominantly ER-localized, a portion may exist elsewhere, suggesting potential moonlighting functions that warrant careful methodological consideration .

How can researchers differentiate between direct and indirect effects of ENPL-1 on insulin secretion?

Differentiating between direct and indirect effects of ENPL-1 on insulin secretion requires careful experimental design:

• Temporal control systems: Implement heat-shock inducible or drug-inducible ENPL-1 expression to observe immediate versus delayed effects on insulin secretion.

• Domain-specific mutations: Generate ENPL-1 variants with mutations in specific functional domains to separate different activities:

  • Client binding domain mutations to disrupt proinsulin interaction

  • ATPase domain mutations to affect chaperone activity

  • ER retention signal modifications to alter localization

• Direct binding assays: Demonstrate physical interaction between ENPL-1 and proinsulin/pro-DAF-28 through techniques like FRET, BiFC, or proximity ligation assays in live cells.

• Rescue experiments with domain specificity: Test whether wild-type ENPL-1 or domain-specific mutants can rescue insulin secretion defects in enpl-1 mutants.

• ER stress uncoupling experiments: Use chemical chaperones (like 4-PBA) or XBP-1 overexpression to alleviate ER stress independently of ENPL-1 function, determining whether insulin secretion defects persist.

Research has shown that ENPL-1 binds directly to proinsulin/pro-DAF-28 via its client binding domain and is both necessary and sufficient for proper insulin secretion. When overexpressed, ENPL-1 increases DAF-28::GFP/insulin accumulation in coelomocytes, suggesting a direct role in the secretion process rather than just an indirect effect through general ER homeostasis .

What are the optimal protocols for immunoprecipitation using ENPL-1 antibodies?

For optimal immunoprecipitation of ENPL-1 and its interaction partners:

• Sample preparation considerations:

  • Use gentle lysis buffers containing 1% NP-40 or 0.5% Triton X-100, 150mM NaCl, 50mM Tris-HCl (pH 7.4), and protease inhibitors

  • Include appropriate phosphatase inhibitors if studying phosphorylation-dependent interactions

  • Consider crosslinking approaches (e.g., DSP or formaldehyde) for capturing transient interactions

• Antibody selection and validation:

  • Validate antibody specificity using enpl-1 mutant strains as negative controls

  • For tagged ENPL-1 (e.g., 3xFlag::ENPL-1), commercial anti-tag antibodies often provide higher specificity than custom antibodies

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

• IP procedure optimization:

  • Use a ratio of 2-5μg antibody per 500μg of total protein

  • Incubate overnight at 4°C with gentle rotation

  • Perform at least 3-5 washes with buffer containing reduced detergent concentration

• Controls and validation:

  • Include IgG control immunoprecipitations

  • Validate interactions through reciprocal IP experiments

  • Confirm specificity using enpl-1 mutants or knockdown conditions

• Analysis of co-immunoprecipitated proteins:

  • Western blotting for known or suspected interactors

  • Mass spectrometry for unbiased identification of binding partners

Research using ENPL-1 immunoprecipitation has successfully demonstrated its interaction with proinsulin/pro-DAF-28, confirming the role of ENPL-1 in insulin processing pathways .

What considerations are important when using cryoEM approaches for antibody characterization?

While traditional antibody characterization methods involve extensive screening, newer cryoEM-based approaches offer several advantages with important considerations:

• Sample preparation for cryoEM:

  • Ensure high purity (>95%) of both antibody and antigen preparations

  • Optimize antibody:antigen ratios to achieve uniform complex formation

  • Consider grid types and blotting conditions to achieve thin, uniform ice

• Data collection strategy:

  • Collect sufficient micrographs to ensure adequate particle numbers (typically >100,000 particles)

  • Implement strategies to handle heterogeneity in antibody-antigen complexes

  • Consider tilted data collection to address preferred orientation issues

• Processing considerations:

  • Implement classification approaches to separate different binding modes

  • Use local refinement techniques to maximize resolution at the antibody-antigen interface

  • Apply methods like cryoEMPEM (cryoEM Polyclonal Epitope Mapping) to analyze polyclonal antibody responses

• Model building and validation:

  • Build models carefully, particularly for the variable regions of antibodies

  • Validate using independent datasets or orthogonal techniques

  • Consider tools like ABodyBuilder for initial antibody model generation

• Integration with sequence data:

  • Combine structural information with next-generation sequencing of antibody repertoires

  • Use computational approaches to match observed electron density with candidate sequences

CryoEM approaches can significantly accelerate antibody discovery by providing structural information rapidly, which can then guide sequence identification and further characterization .

How can researchers validate ENPL-1 antibody specificity in immunohistochemistry applications?

Validating ENPL-1 antibody specificity for immunohistochemistry requires comprehensive controls and cross-verification:

• Essential negative controls:

  • enpl-1 null mutants (enpl-1(ok1964) or enpl-1(tm3738)) as biological negative controls

  • Peptide competition assays to demonstrate binding specificity

  • Secondary antibody-only controls to assess background staining

• Positive controls and pattern verification:

  • Compare staining patterns with ENPL-1::sfGFP or 3xFlag::ENPL-1 expression patterns

  • Verify co-localization with established ER markers (e.g., HSP-4/BIP)

  • Compare with in situ hybridization data for enpl-1 mRNA

• Cross-validation with multiple antibodies:

  • Use antibodies targeting different epitopes of ENPL-1

  • Compare polyclonal and monoclonal antibody staining patterns

  • Verify with antibodies against epitope-tagged ENPL-1 variants

• Fixation and permeabilization optimization:

  • Test multiple fixation protocols (e.g., paraformaldehyde, methanol, Bouin's)

  • Optimize antigen retrieval methods if necessary

  • Determine optimal permeabilization conditions for accessing ER lumen epitopes

• Quantitative validation:

  • Implement quantitative image analysis to compare staining intensities across genotypes

  • Correlate with Western blot quantification from the same tissues/samples

Research has established that ENPL-1 is broadly expressed with particularly strong expression in neurons, vulva, germline, and intestine, presenting a perinuclear pattern characteristic of ER localization. These known patterns provide a reference for validating new antibodies .

How should researchers interpret changes in ENPL-1 levels in the context of ER stress experiments?

Interpreting changes in ENPL-1 levels during ER stress requires careful analysis:

• Baseline considerations:

  • Establish normal ENPL-1 expression levels across tissues and developmental stages

  • Determine relationship between ENPL-1 and canonical ER stress markers (HSP-4/BIP, XBP-1s)

  • Consider post-translational modifications that may affect antibody recognition

• Experimental design for accurate interpretation:

  • Include time-course analyses to distinguish between early and late ER stress responses

  • Compare multiple ER stress inducers (tunicamycin, thapsigargin, DTT) for pathway specificity

  • Distinguish between transcriptional and post-transcriptional regulation using RT-qPCR and Western blot

• Analytical framework:

  • Normalize ENPL-1 levels to appropriate housekeeping genes/proteins

  • Consider ratiometric analysis of ENPL-1 to other ER chaperones

  • Implement statistical approaches that account for biological variability

• Integration with functional readouts:

  • Correlate ENPL-1 levels with functional measures such as insulin secretion capacity

  • Assess relationship between ENPL-1 levels and phenotypic outcomes (growth, fertility)

  • Evaluate impact on DAF-28/insulin processing and localization

Research has shown that in enpl-1(ok1964) mutants, expression of the ER stress marker hsp-4/BIP is significantly upregulated compared to wild type. This elevated ER stress correlates with accumulation of DAF-28::GFP in neuronal cell bodies and inhibition of insulin secretion, demonstrating the complex relationship between ENPL-1, ER stress, and functional outcomes .

What approaches can differentiate between ENPL-1 and related ER chaperones in functional studies?

Differentiating ENPL-1 from other ER chaperones requires targeted approaches:

• Genetic separation strategies:

  • Generate specific loss-of-function mutations in individual chaperones

  • Create double/triple mutants to assess functional redundancy

  • Implement tissue-specific or temporally controlled knockdown/knockout approaches

• Biochemical differentiation:

  • Use co-immunoprecipitation with client-specific antibodies to identify distinct binding partners

  • Perform in vitro binding assays with purified proteins to establish direct interactions

  • Analyze ATP binding and hydrolysis to distinguish mechanistic differences

• Client specificity analysis:

  • Assess binding specificity for DAF-28/insulin versus other secreted proteins

  • Compare effects on trafficking and secretion of multiple cargo proteins

  • Map binding sites through domain swap experiments between chaperones

• Chaperone network analysis:

  • Map genetic and physical interactions between different chaperones

  • Perform epistasis analysis placing chaperones in hierarchical or parallel pathways

  • Use proximity labeling approaches (BioID, APEX) to identify chaperone-specific interactomes

• Response kinetics:

  • Compare temporal dynamics of different chaperones during ER stress

  • Assess recovery kinetics following stress resolution

  • Analyze age-dependent changes in chaperone function and expression

Research has established that ENPL-1 has specific roles in insulin processing and secretion that may not be redundant with other chaperones. The ability of ENPL-1 to bind proinsulin/pro-DAF-28 through its client binding domain suggests a specialized function distinct from general chaperone activities .

What considerations are important when designing rescue experiments using ENPL-1 transgenes?

Designing effective rescue experiments with ENPL-1 transgenes requires attention to multiple experimental parameters:

• Transgene design considerations:

  • Expression level control: Use endogenous promoter and 3'UTR to maintain physiological expression

  • Tagging strategies: Consider tag position (N- vs. C-terminal) and size to minimize functional interference

  • Include introns and regulatory elements that may affect expression timing and levels

• Integration strategies:

  • Single-copy integration (e.g., MosSCI) to avoid overexpression artifacts

  • CRISPR knock-in approaches for endogenous locus modification

  • Site-specific integration to minimize position effects

• Experimental validation requirements:

  • Quantify transgene expression at mRNA and protein levels relative to endogenous expression

  • Verify proper localization using subcellular fractionation or imaging

  • Confirm functional activity through biochemical assays

• Comprehensive phenotypic assessment:

  • Evaluate multiple phenotypes (e.g., insulin secretion, fertility, body size, cisplatin sensitivity)

  • Quantify rescue efficiency across different tissues and developmental stages

  • Assess rescue under both standard and stress conditions

• Control experiments:

  • Include domain mutants as specificity controls

  • Test rescue with orthologs (e.g., human GRP94) to assess functional conservation

  • Use inactive mutants (e.g., ATPase-dead variants) as negative controls

Research has demonstrated that expression of ENPL-1 from both its endogenous locus together with a transgene-derived protein fully rescued multiple phenotypes in enpl-1 mutants, including cisplatin sensitivity and sterility. Additionally, ENPL-1 overexpression was sufficient to increase DAF-28::GFP/insulin accumulation in coelomocytes, confirming the functional specificity of the rescue .

How can ENPL-1 research in C. elegans inform studies of GRP94 in mammalian insulin processing?

ENPL-1 research in C. elegans provides valuable insights for mammalian GRP94 studies through several translational approaches:

• Conserved mechanism identification:

  • Compare binding domains and interaction specificities between ENPL-1 and GRP94

  • Assess whether client selectivity mechanisms are conserved across species

  • Determine if ATP dependency of chaperone function follows similar patterns

• Disease-relevant phenotypic models:

  • Utilize C. elegans as a rapid screening platform for mutations identified in human GRP94

  • Assess functional conservation through rescue experiments with human GRP94 in enpl-1 mutants

  • Model diabetes-related phenotypes through manipulation of insulin processing and secretion

• Drug discovery applications:

  • Screen for compounds that modulate ENPL-1 function in C. elegans

  • Test whether compounds affecting GRP94 show similar effects on ENPL-1

  • Identify conserved small molecule binding sites for targeted drug development

• Pathway conservation analysis:

  • Map genetic interactions in C. elegans and test conservation in mammalian systems

  • Compare consequences of chaperone dysfunction on insulin production and secretion

  • Assess similar roles in ER stress responses across species

Research has established that ENPL-1 promotes insulin secretion in C. elegans via regulation of proinsulin processing, similar to the role of GRP94 in mammalian systems. The binding between ENPL-1 and proinsulin/pro-DAF-28 via a specific domain mirrors interaction mechanisms in higher organisms, suggesting that findings in C. elegans may translate to mammalian insulin processing pathways .

What methods can be used to develop and validate ENPL-1-specific antibodies for cross-species applications?

Developing cross-species ENPL-1/GRP94 antibodies requires strategic epitope selection and comprehensive validation:

• Epitope selection strategies:

  • Identify highly conserved regions between C. elegans ENPL-1 and mammalian GRP94

  • Target functional domains with structural conservation

  • Avoid regions with high sequence similarity to other HSP90 family members

  • Consider evolutionary conservation across multiple species for broader applicability

• Antibody development approaches:

  • Generate monoclonal antibodies against conserved epitopes

  • Produce domain-specific antibodies targeting functional regions

  • Develop conformation-specific antibodies that recognize active states

• Cross-species validation pipeline:

  • Test antibodies against recombinant proteins from multiple species

  • Validate in knockout/knockdown systems for each target species

  • Perform epitope mapping to confirm binding to intended conserved regions

  • Compare with established species-specific antibodies where available

• Application-specific validation:

  • Validate for multiple techniques (Western blot, IP, IHC, ELISA)

  • Optimize conditions for each species and application

  • Determine sensitivity and specificity metrics for each application

• Controls for cross-reactivity:

  • Test against related HSP90 family members

  • Validate in systems with multiple knockout/knockdowns

  • Perform peptide competition assays with species-specific peptides

Cross-species antibodies would facilitate comparative studies between C. elegans ENPL-1 and mammalian GRP94, enabling direct translation of findings from model organisms to mammalian systems in the context of insulin processing, ER stress responses, and related pathologies .

How can structural analysis of ENPL-1-antibody complexes inform epitope mapping strategies?

Structural analysis of ENPL-1-antibody complexes provides powerful insights for epitope mapping through several advanced approaches:

• CryoEM-based epitope mapping workflow:

  • Prepare ENPL-1-antibody complexes for single-particle cryoEM analysis

  • Process data using advanced classification to resolve heterogeneity

  • Generate 3D reconstructions at near-atomic resolution (3-4Å)

  • Build atomic models into density maps to identify specific epitope-paratope contacts

• Integration with sequence-based methods:

  • Combine structural information with next-generation sequencing data of antibody repertoires

  • Apply computational algorithms to match observed structural features with candidate sequences

  • Use structure-guided sequence assignment to identify antibodies binding specific epitopes

• Comparative epitope analysis:

  • Map epitopes across evolutionary related proteins (ENPL-1 vs. GRP94)

  • Identify conserved epitopes that may target functionally important regions

  • Compare epitope accessibility in different conformational states

• Application to polyclonal responses:

  • Use cryoEMPEM (cryoEM Polyclonal Epitope Mapping) to analyze complex polyclonal samples

  • Classify particles based on binding locations to generate epitope maps

  • Quantify relative abundance of antibodies targeting different epitopes

• Functional correlation analyses:

  • Correlate epitope locations with functional domains of ENPL-1

  • Identify neutralizing vs. non-neutralizing epitopes based on functional assays

  • Map epitopes that affect specific protein-protein interactions

Modern structural biology approaches like cryoEM can provide direct visualization of antibody-antigen complexes at near-atomic resolution, allowing precise epitope mapping without the need for extensive mutagenesis studies. Such approaches can significantly accelerate antibody characterization and inform the development of more specific reagents for ENPL-1 research .

What are common pitfalls in ENPL-1 protein detection and how can they be addressed?

Detecting ENPL-1 protein can present several challenges that require specific troubleshooting approaches:

• Protein extraction challenges:

  • Problem: Incomplete extraction due to ER localization

  • Solution: Use extraction buffers containing 1-2% SDS or 8M urea to ensure complete solubilization

  • Validation: Compare multiple extraction methods with known ER protein controls

• Antibody specificity issues:

  • Problem: Cross-reactivity with other HSP90 family members

  • Solution: Validate antibody specificity using enpl-1 mutants as negative controls

  • Alternative: Use epitope-tagged ENPL-1 with commercial tag antibodies for higher specificity

• Detection sensitivity limitations:

  • Problem: Low abundance in certain tissues

  • Solution: Implement sample enrichment through immunoprecipitation before Western blotting

  • Alternative: Use more sensitive detection methods like chemiluminescence or fluorescent secondary antibodies

• Post-translational modifications:

  • Problem: Modifications affecting antibody recognition

  • Solution: Use multiple antibodies targeting different epitopes

  • Analysis: Include phosphatase or deglycosylation treatments to assess modification impact

• Size verification challenges:

  • Problem: Unexpected migration patterns on SDS-PAGE

  • Solution: Include known molecular weight markers and positive controls

  • Validation: Confirm identity through mass spectrometry when possible

Research on ENPL-1 has successfully employed Western blotting to confirm the absence of protein expression in enpl-1(ok1964) and enpl-1(tm3738) mutants, as well as to validate the increased expression in transgenic rescue lines, demonstrating that these technical challenges can be overcome with appropriate methodology .

How can researchers optimize immunohistochemistry protocols for ENPL-1 in different C. elegans tissues?

Optimizing immunohistochemistry for ENPL-1 across different C. elegans tissues requires tissue-specific adaptations:

• Tissue-specific fixation strategies:

  • Neurons: Use 2-4% paraformaldehyde with shorter fixation times (15-30 minutes)

  • Germline: Methanol fixation at -20°C for better preservation of germline structure

  • Intestine: Test a combination of paraformaldehyde and glutaraldehyde for improved membrane preservation

  • Embryos: Use freeze-crack methods followed by methanol fixation

• Permeabilization optimization:

  • Problem: Insufficient antibody access to ER lumen

  • Solution: Test graduated series of Triton X-100 concentrations (0.1-1%)

  • Alternative: For challenging tissues, try freeze-thaw cycles or limited protease digestion

• Background reduction strategies:

  • Problem: High autofluorescence, especially in intestine

  • Solution: Include extended blocking (5% BSA, 5% goat serum, 0.05% Tween-20)

  • Alternative: Use Sudan Black B treatment to reduce lipofuscin autofluorescence

• Signal amplification approaches:

  • Problem: Weak signal in tissues with lower expression

  • Solution: Implement tyramide signal amplification systems

  • Alternative: Use secondary antibody conjugated to bright fluorophores (Alexa Fluor 488/555/647)

• Visualization techniques:

  • Problem: Distinguishing ENPL-1 from other ER proteins

  • Solution: Use co-staining with established ER markers with spectrally distinct fluorophores

  • Analysis: Apply deconvolution algorithms to improve signal-to-noise ratio

Research has established that ENPL-1 is broadly expressed with particularly strong signals in neurons, vulva, germline, and intestine. By optimizing protocols for each specific tissue, researchers can achieve more consistent and reliable immunohistochemistry results for ENPL-1 detection .

What strategies can overcome challenges in co-immunoprecipitation of ENPL-1 with client proteins?

Co-immunoprecipitation of ENPL-1 with its client proteins presents several challenges that can be addressed through specific optimization strategies:

• Preserving transient interactions:

  • Challenge: ENPL-1-client interactions may be transient or ATP-dependent

  • Solution: Use chemical crosslinking (DSP, formaldehyde) before lysis

  • Alternative: Include ATP/ADP in buffers to stabilize specific conformational states

• Buffer optimization for complex stability:

  • Challenge: Standard IP buffers may disrupt ENPL-1-client complexes

  • Solution: Test buffers with varying ionic strength (100-300mM NaCl)

  • Optimization: Include glycerol (10%) and mild detergents (0.5% NP-40 or 0.1% digitonin)

• Client-specific detection challenges:

  • Challenge: Low abundance of processed insulin forms

  • Solution: Use tagged insulin variants (DAF-28::GFP) for enhanced detection

  • Alternative: Employ mass spectrometry for unbiased identification of co-precipitated proteins

• Controlling for non-specific binding:

  • Challenge: Abundant ER proteins may co-precipitate non-specifically

  • Solution: Include stringent controls (IgG, unrelated ER proteins)

  • Validation: Perform reciprocal IPs using antibodies against client proteins

• Distinguishing direct from indirect interactions:

  • Challenge: Co-precipitated proteins may interact indirectly via larger complexes

  • Solution: Use purified recombinant proteins in vitro to test direct interactions

  • Alternative: Implement proximity labeling approaches (BioID, APEX) in vivo