CAND1 Antibody, HRP conjugated

What is CAND1 and why is it important in cellular biology?

CAND1 functions as a key assembly factor of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complexes, promoting the exchange of substrate-recognition F-box subunits in SCF complexes. It acts as an F-box protein exchange factor, playing a critical role in establishing the cellular repertoire of SCF complexes. CAND1's exchange activity is coupled with cycles of neddylation conjugation: in the deneddylated state, CAND1 binds to CUL1-RBX1, increasing dissociation of the SCF complex and promoting exchange of the F-box protein. This function extends to other cullin-RING E3 ubiquitin ligase complexes as well, though with varying effects depending on the specific cullin family member involved.

How does CAND1 interact differently with various Cullin-RING ligase systems?

Unexpectedly, CAND1 shows differential regulation of CRL complexes. While CAND1 activates CRL1s by promoting the exchange of diverse CUL1 substrate receptor modules in human cells, allowing "just-in-time" recruitment for target protein ubiquitination, it inhibits CRL2-mediated protein degradation. This inhibitory effect on CRL2 arises from altered binding kinetics - CAND1 dramatically increases the dissociation rate of CRL2s but hardly accelerates the assembly of stable CRL2s. This differential regulation appears to help distinguish target proteins with different affinities for CRL2s, presenting a mechanism for selective protein degradation with proper pacing in the cellular environment.

What is the structural basis for CAND1 interactions with Cullin proteins?

CAND1 exhibits a horseshoe-shaped structure that clamps around cullins, particularly CUL1, and buries the neddylation site. This structural arrangement prevents neddylation of CAND1-bound CUL1, and conversely, CAND1 cannot stably bind to neddylated CUL1. When CAND1 associates with unneddylated CUL1 in a CRL1 complex, a CAND1-SKP1 adaptor clash catalyzes replacement of the substrate receptor modules assembled on the same CUL1 scaffold. This structural interaction defines CAND1's role as an exchange factor for CRL1s. CAND1 also interacts with other cullin family members including CUL2, CUL3, CUL4, and CUL5, suggesting a broader role in regulating various CRLs, though with differing functional outcomes.

What are the critical considerations when selecting a CAND1 antibody for research applications?

When selecting a CAND1 antibody, researchers should consider: (1) The specific epitope targeted - antibodies targeting different regions of CAND1 may yield different results depending on protein interactions and conformational changes; (2) Antibody format - monoclonal versus polyclonal, with monoclonals offering higher specificity while polyclonals may provide stronger signals; (3) Species reactivity - ensure compatibility with your experimental system; (4) Validated applications - confirm the antibody has been tested for your specific application (WB, IP, IHC, ICC, etc.); (5) Clone specificity - different clones may have distinct binding properties even when targeting the same region. For example, the EPR14242(B) clone is a rabbit recombinant monoclonal antibody targeting the C-terminal region of CAND1, suitable for multiple applications including IP, WB, ICC/IF, Flow Cytometry, and IHC-P, with validated reactivity for human samples.

How can researchers validate CAND1 antibody specificity for their experimental systems?

Validating antibody specificity is crucial for reliable research results. Recommended validation approaches include: (1) Positive and negative control samples - using tissues/cells known to express or lack CAND1; (2) Knockdown/knockout validation - comparing antibody reactivity in wild-type versus CAND1 knockdown/knockout samples; (3) Peptide competition assays - pre-incubating the antibody with the immunizing peptide should block specific binding; (4) Multiple antibody comparison - using different antibodies targeting distinct epitopes of CAND1 should yield consistent patterns; (5) Molecular weight verification - confirming detection at the expected molecular weight (~120 kDa for CAND1); (6) Cross-reactivity testing - especially important when working with antibodies claimed to work across multiple species. For CAND1 studies, knockout validation is particularly informative, as demonstrated in studies using CAND1/CAND2 double knockout (DKO) HEK293 cells to confirm antibody specificity and analyze CAND1 function.

What are the optimal dilutions and conditions for using CAND1 antibodies in different applications?

While optimal conditions vary by antibody clone and application, general guidelines based on available CAND1 antibodies include:

For Western blotting of CAND1, researchers should consider using gradient gels (4-12%) to accommodate its high molecular weight (~120 kDa). Validation steps should include testing multiple dilutions to determine optimal signal-to-noise ratio for each specific experimental system.

What are the most effective methods for studying CAND1-Cullin interactions using antibody-based approaches?

To effectively study CAND1-Cullin interactions, researchers can employ several antibody-based approaches:

• Co-immunoprecipitation (Co-IP): This is particularly effective for detecting native protein complexes. IP CAND1 and blot for cullins or vice versa. This approach has successfully demonstrated that CAND1 preferentially binds unneddylated CUL2 and that CUL2 does not form a stable complex with both CAND1 and the VHL- ELOB- ELOC (VBC) substrate receptor module.

• Immunofluorescence co-localization: Use differentially labeled antibodies against CAND1 and cullins to visualize their co-localization in cells under different conditions, such as before and after treatment with neddylation inhibitors like MLN4924.

• Proximity ligation assay (PLA): This technique can detect protein interactions with high sensitivity and specificity, allowing visualization of CAND1-cullin interactions in situ.

• FRET/BRET analysis: When combined with fluorescently tagged proteins, antibodies can help validate the proximity measurements obtained.

• GST-pulldown assays: Using recombinant GST-CAND1 to pull down cullins from cell lysates followed by detection with specific antibodies has successfully identified cullin family members (Cul1, Cul2, Cul3, Cul4, and Cul5) and ROC1 as CAND1 binding partners.

These methods have revealed that CAND1 interacts with multiple cullins but with different functional outcomes, activating CRL1/3/4s while inhibiting CRL2s.

How can researchers effectively use CAND1 antibodies to study the neddylation and deneddylation cycle of Cullins?

To study the neddylation cycle using CAND1 antibodies, researchers should:

• Use neddylation inhibitors: Treat cells with MLN4924 to block neddylation, then analyze CAND1-cullin interactions by co-IP and Western blot. This approach has demonstrated that CAND1 preferentially binds unneddylated CUL2.

• Sequential immunoprecipitation: IP for CAND1 followed by Western blot for cullins and NEDD8 to determine the neddylation status of CAND1-bound cullins.

• Time-course experiments: Analyze CAND1-cullin interactions at different time points after adding or removing neddylation inhibitors to track dynamic changes.

• COP9 signalosome manipulation: As CSN regulates CAND1 activity by controlling deneddylation, researchers can manipulate CSN components and observe effects on CAND1-cullin interactions.

• SCCRO/DCUN1D1 studies: Include analysis of SCCRO, which is an essential component of the neddylation E3 complex and involved in releasing inhibitory effects of CAND1 on CRL assembly and activity.

These approaches can help track the cycling of cullins between CAND1-bound inactive states and neddylated active states, providing insights into the regulation of CRL activities in different cellular contexts.

What protocols yield optimal results for detecting CAND1 in different cellular compartments?

For optimal detection of CAND1 in different cellular compartments:

• Subcellular fractionation protocol:

  • Lyse cells in hypotonic buffer (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, protease inhibitors)

  • Separate nuclei by centrifugation (1,300 g, 5 min, 4°C)

  • Further fractionate cytoplasm into soluble and membrane fractions

  • Extract nuclear proteins with high-salt buffer

  • Analyze fractions by Western blot with CAND1 antibody

• Immunofluorescence optimization:

  • Fixation: 4% paraformaldehyde (10 min) or -20°C acetone (5 min) - the latter has been successfully used for CAND1 detection in HeLa cells

  • Permeabilization: 0.2% Triton X-100 in PBS (10 min)

  • Blocking: 3% BSA in PBS (1 hour)

  • Primary antibody: Incubate with anti-CAND1 at 1:200-1:500 dilution overnight at 4°C

  • Secondary antibody: Fluorophore-conjugated anti-rabbit IgG

  • Counterstain nuclei with DAPI

• Proximity to cullins:

  • Use dual immunofluorescence staining for CAND1 and cullins

  • Analyze co-localization in different cellular compartments using confocal microscopy

These protocols should be optimized for specific antibodies and cell types, with appropriate controls to confirm specificity of the observed signals.

What are common pitfalls in CAND1 antibody experiments and how can they be addressed?

Common pitfalls and solutions in CAND1 antibody experiments include:

• Cross-reactivity issues: CAND1 shares some sequence homology with CAND2/TIP120B. To address this:

  • Use antibodies specifically validated against both CAND1 and CAND2

  • Include CAND1/CAND2 double knockout controls when possible

  • Perform peptide competition assays to confirm specificity

• Neddylation state interference: CAND1 preferentially binds unneddylated cullins, which can affect experimental outcomes.

  • Include neddylation inhibitor controls (MLN4924)

  • Compare results with and without deneddylation treatments

• Extraction difficulties: CAND1-cullin complexes may be incompletely extracted.

  • Optimize lysis conditions (detergent type/concentration, salt concentration)

  • Try different extraction methods for nuclear vs. cytoplasmic fractions

• Antibody binding affected by protein interactions: CAND1's horseshoe shape around cullins may obscure epitopes.

  • Use antibodies targeting different regions of CAND1

  • Consider native vs. denaturing conditions depending on experimental goals

• Signal variability in immunostaining: CAND1 distribution may vary with cell cycle or stress.

  • Synchronize cells when possible

  • Include appropriate cellular markers to categorize cells by state

These challenges can be addressed through careful experimental design and appropriate controls to ensure reliable interpretation of results.

How should researchers interpret contradictory results between different CAND1 antibody-based experiments?

When facing contradictory results with different CAND1 antibodies, researchers should:

• Evaluate epitope locations: Different antibodies may detect distinct pools of CAND1 based on epitope accessibility. The EPR14242(B) clone targets the C-terminal region, while other antibodies may target different domains. Compare the epitope locations with known CAND1 interaction interfaces.

• Consider cullin-specific effects: CAND1 has opposing effects on different CRL complexes (activating CRL1/3/4 while inhibiting CRL2). Contradictory results may reflect these biological differences rather than technical issues.

• Assess neddylation status: CAND1 preferentially interacts with unneddylated cullins. Differences in neddylation status across experiments could cause contradictory results. Include MLN4924 controls to standardize neddylation states.

• Review experimental conditions: Buffer composition, detergents, salt concentration, and incubation times can all affect CAND1-cullin interactions and antibody binding.

• Validate with orthogonal methods: Combine antibody-based approaches with other techniques like mass spectrometry, proximity labeling, or functional assays to resolve contradictions.

• Consider cell type and context: CAND1 function may vary across cell types or conditions. The recent finding that CAND1 inhibits CRL2 but activates other CRLs highlights the importance of cellular context.

By systematically analyzing these factors, researchers can reconcile apparently contradictory results and gain deeper insights into CAND1 biology.

What controls are essential when using CAND1 antibodies in complex experimental systems?

Essential controls for CAND1 antibody experiments include:

• Genetic controls:

  • CAND1 knockout or knockdown cells (ideally CAND1/CAND2 double knockout to eliminate potential compensation)

  • Rescue experiments with CAND1 re-expression

  • These controls have been successfully employed in studies demonstrating CAND1's inhibitory effect on CRL2-mediated protein degradation

• Biochemical controls:

  • Neddylation status controls (±MLN4924 treatment)

  • Peptide competition assays to confirm antibody specificity

  • Isotype controls for immunoprecipitation experiments

  • Secondary antibody-only controls for immunostaining

• Sample preparation controls:

  • Total protein loading controls (housekeeping proteins)

  • Subcellular fraction purity markers when analyzing compartment-specific distribution

  • Time course controls for dynamic processes

• Cross-validation controls:

  • Multiple antibodies targeting different CAND1 epitopes

  • Orthogonal detection methods (e.g., tagged CAND1 expression)

  • Protein-protein interaction verification through reciprocal co-IPs (e.g., IP for CAND1 and blot for cullins, then IP for cullins and blot for CAND1)

These controls help ensure that observed results genuinely reflect CAND1 biology rather than technical artifacts or antibody non-specificity.

How can CAND1 antibodies be used to study the differential regulation of distinct Cullin-RING ligase complexes?

CAND1 antibodies can be powerful tools for investigating its differential regulation of CRL complexes through several advanced approaches:

• Differential co-IP analysis: Perform CAND1 immunoprecipitation followed by blotting for different cullins to compare binding affinities and dynamics. This approach has revealed that CAND1 preferentially binds unneddylated CUL2, similar to its interaction with CUL1, despite having opposing functional effects.

• Dynamic interaction studies: Use CAND1 antibodies in FRAP (Fluorescence Recovery After Photobleaching) or live-cell imaging coupled with fluorescently tagged cullins to analyze the kinetics of CAND1-cullin interactions in real-time.

• Substrate fate tracking: Combine CAND1 antibodies with antibodies against CRL substrates to monitor how CAND1 manipulation affects substrate degradation differentially across CRL types. This approach has shown that CAND1 inhibits ARV-771-induced degradation of BRD4 (a CRL2 VHL neo-substrate) while it accelerates degradation of CRL1 substrates.

• Proximity-dependent labeling: Use CAND1 antibodies to validate BioID or APEX2-based proximity labeling results, helping map the differential interactome of CAND1 with various CRL components.

• Structural studies validation: CAND1 antibodies can be used to confirm structural models of CAND1-cullin interactions through techniques like crosslinking mass spectrometry or hydrogen-deuterium exchange.

These approaches can help elucidate how CAND1 differentially regulates distinct CRL complexes, with potential implications for developing targeted therapeutic strategies.

What methodologies can researchers employ to study CAND1's role in PROTAC-mediated protein degradation?

PROteolysis TArgeting Chimeras (PROTACs) represent an important new class of therapeutic compounds that co-opt E3 ligases (often CRL2 VHL) to target specific proteins for degradation. To study CAND1's role in PROTAC function:

• Cellular degradation kinetics analysis:

  • Compare PROTAC efficacy in wild-type vs. CAND1 knockout cells

  • Use pulse-chase experiments with CAND1 antibody detection to monitor substrate degradation rates

  • Recent research has shown increased elimination of BRD2 by the PROTAC ARV-771 in CAND1/CAND2 double knockout cells compared to wild-type cells, with physiologically significant consequences including increased apoptosis in the knockout cells

• PROTAC affinity modulation studies:

  • Use different PROTACs with varying affinities for CRL2 VHL to analyze how CAND1 affects selective protein degradation

  • CAND1's inhibitory effect on CRL2 VHL helps distinguish target proteins with different affinities, presenting a mechanism for selective protein degradation

• CRL2 assembly/disassembly dynamics:

  • Use CAND1 antibodies in conjunction with VHL, ELOB, ELOC antibodies to monitor how PROTACs affect CRL2 complex stability

  • Analyze how CAND1 overexpression or depletion affects the kinetics of PROTAC-induced CRL2 recruitment to substrates

• Neddylation cycle modulation:

  • Combine MLN4924 (neddylation inhibitor) with CAND1 manipulation to determine how the neddylation cycle influences PROTAC efficacy

  • Monitor cullin neddylation status in response to PROTACs with and without CAND1

These approaches can provide crucial insights into optimizing PROTAC design and understanding the factors influencing their efficacy and selectivity.

What are the most sophisticated imaging techniques for visualizing CAND1-cullin interactions in live cells?

Advanced imaging techniques for visualizing CAND1-cullin interactions include:

• Förster Resonance Energy Transfer (FRET):

  • Tag CAND1 and cullins with appropriate donor/acceptor fluorophores

  • Use CAND1 antibodies to validate FRET signals in fixed cells

  • Monitor real-time interaction changes following treatments affecting neddylation

• Bioluminescence Resonance Energy Transfer (BRET):

  • Similar to FRET but using luciferase-fluorophore pairs for reduced phototoxicity

  • Particularly useful for long-term monitoring of CAND1-cullin interactions

• Fluorescence Lifetime Imaging Microscopy (FLIM):

  • Measure changes in fluorescence lifetime of donor fluorophores when CAND1-cullin interactions occur

  • Less susceptible to concentration artifacts than intensity-based FRET

• Single Molecule Tracking:

  • Use quantum dot-conjugated antibodies against CAND1 for long-term tracking

  • Analyze diffusion coefficients to identify bound vs. unbound states

• Lattice Light Sheet Microscopy with Adaptive Optics:

  • Provides high spatiotemporal resolution with reduced phototoxicity

  • Use fluorescent protein-tagged CAND1 and cullins, validated with antibody staining

• Expansion Microscopy:

  • Physically expand samples to improve spatial resolution of CAND1-cullin complexes

  • Use CAND1 antibodies optimized for the expanded sample conditions

These advanced techniques can reveal the dynamic nature of CAND1-cullin interactions at unprecedented resolution, helping elucidate the spatial and temporal regulation of CRL assembly and substrate processing in living cells.