CDC48E Antibody

What is CDC48 and what cellular functions does it perform?

CDC48 is a ubiquitin-dependent molecular chaperone that mediates various degradative and regulatory processes in cells. In humans, it's known as valosin-containing protein (VCP), an 806-amino acid protein that plays crucial roles in the fragmentation of Golgi stacks during mitosis and their reassembly after mitosis. CDC48 forms a complex with p47 and the ubiquitin fusion degradation 1 (Ufd1)-nuclear protein localization 4 (Npl4) heterodimer, which is involved in binding polyubiquitinated substrates at the cytoplasmic face of the endoplasmic reticulum (ER) membrane and transferring them to the 26S proteasome. The protein is localized to the nucleus, ER, and cytoplasm, features phosphorylated post-translational modifications, and is widely expressed across many tissue types .

What is the significance of CDC48 in neurodevelopment?

CDC48 plays an essential protective role during neurodevelopment. Studies using zebrafish embryos demonstrated that CDC48 deficiency produces lethal embryonic phenotypes, including defects in neuronal outgrowth and neurodegeneration. When CDC48 is knocked down, polyubiquitinated proteins accumulate in the inner plexiform and ganglion cell layers, as well as the diencephalon and mesencephalon, indicating that the degradation of polyubiquitinated proteins by the ubiquitin-proteasome system (UPS) is blocked. These abnormal phenotypes can be rescued by CDC48 or human valosin-containing protein overexpression, demonstrating that the protective function of CDC48 is essential for proper neurodevelopment and survival .

How are CDC48 antibodies primarily used in research?

CDC48 antibodies are used for antigen-specific immunodetection in biological samples across multiple applications:

• Western Blot (WB): For detecting and quantifying CDC48 protein in cell or tissue lysates, typically using dilutions between 1:500-1:5,000

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of CDC48, with some antibodies optimized for dilutions as high as 1:128,000

• Immunohistochemistry (IHC): For visualizing CDC48 localization in tissue sections and determining its distribution across different cell types

These applications enable researchers to investigate CDC48's involvement in cellular processes, protein degradation pathways, and developmental mechanisms .

What criteria should guide the selection of a CDC48 antibody for specific research applications?

When selecting a CDC48 antibody for experimental use, researchers should evaluate several critical parameters:

For instance, the CDC48 antibody described in search result is a polyclonal antibody from goat that targets an internal region (amino acids 128-142) with the sequence C-PIADTIEGITGNLFD, has been validated for P-ELISA, WB, and IHC applications, and recognizes human CDC48 .

What are optimal protocols for detecting CDC48 using Western blotting?

For reliable Western blot detection of CDC48, researchers should follow this methodological workflow:

• Sample preparation:

  • Dechorionate embryos or lyse cells in an appropriate buffer containing protease inhibitors

  • Include phosphatase inhibitors as CDC48 features phosphorylated modifications

• Protein separation:

  • Use 7-10% SDS-PAGE gels (CDC48/VCP is approximately 97 kDa)

  • Load 20-50 μg of total protein per lane

• Protein transfer and detection:

  • Transfer to PVDF or nitrocellulose membrane

  • Block with 5% non-fat milk or BSA

  • Incubate with anti-CDC48 antibody (typically 1:2500 dilution for zebrafish studies)

  • Use appropriate secondary antibodies (e.g., Alexa Fluor 488 or 596-conjugated)

  • Detect signals using enhanced chemiluminescence

• Controls:

  • Include positive controls (tissues known to express CDC48)

  • Use morpholino oligonucleotides (MO) against CDC48 as negative controls

  • Perform rescue experiments with CDC48 overexpression to confirm specificity

This protocol is adapted from methodology used in zebrafish studies examining CDC48's role in neurodevelopment .

How can researchers validate the specificity of CDC48 antibodies?

Validating CDC48 antibody specificity requires a multi-faceted approach:

• Genetic validation:

  • Use CDC48 knockdown models (e.g., with morpholino oligonucleotides as in zebrafish studies)

  • Compare antibody signal between control and CDC48-depleted samples

  • Verify reduced CDC48 levels (to approximately 12-30% of control levels)

  • Confirm rescue of antibody signal upon CDC48 overexpression

• Epitope competition:

  • Pre-incubate the antibody with immunizing peptide (e.g., C-PIADTIEGITGNLFD)

  • Test whether this pre-incubation abolishes the signal

  • Use a non-related peptide as negative control

• Multiple antibody validation:

  • Compare results using different antibodies targeting distinct CDC48 epitopes

  • For instance, compare antibodies targeting amino acids 128-142 versus 509-521

• Cross-species validation:

  • Test reactivity across species (if antibody is claimed to be cross-reactive)

  • Compare signal patterns in human, yeast, or zebrafish samples as appropriate

This validation strategy ensures that observed signals genuinely represent CDC48 rather than non-specific binding or cross-reactivity .

How can CDC48 antibodies be used to study the ubiquitin-proteasome system in neurodegenerative models?

CDC48 antibodies enable sophisticated analysis of ubiquitin-proteasome system dysfunction in neurodegenerative conditions:

• In vivo bioassay techniques:

  • Introduce fluorescent proteins with polyubiquitination signals into model systems

  • Monitor protein accumulation in neuronal tissues when CDC48 function is compromised

  • Quantify fluorescence in specific brain regions (inner plexiform and ganglion cell layers, diencephalon, mesencephalon)

  • Use CDC48 antibodies to confirm knockdown efficiency

• Double immunostaining:

  • Co-stain tissues with anti-CDC48 and anti-polyubiquitin antibodies

  • Analyze colocalization patterns in normal versus diseased tissues

  • Quantify changes in distribution patterns during disease progression

• Rescue experiments:

  • Test compounds that may restore CDC48 function in diseased models

  • Use CDC48 antibodies to monitor expression levels during treatment

  • Correlate CDC48 restoration with reduction in ubiquitinated protein accumulation

These approaches have revealed that CDC48 deficiency leads to neurodegeneration associated with accumulation of polyubiquitinated proteins, suggesting a protective role for CDC48 that might be leveraged therapeutically .

What methodologies enable investigation of CDC48's role in endoplasmic reticulum-associated degradation (ERAD)?

To study CDC48's function in ERAD, researchers can implement these antibody-dependent methodologies:

• Subcellular fractionation analysis:

  • Separate ER membrane fractions from other cellular components

  • Use CDC48 antibodies to detect CDC48 recruitment to ER membranes

  • Quantify CDC48-associated proteins in the ER fraction under normal and stress conditions

• Immunoprecipitation of CDC48 complexes:

  • Pull down CDC48 using specific antibodies

  • Analyze co-precipitation of ERAD-specific cofactors (p47, Ufd1-Npl4)

  • Monitor changes in complex formation under different cellular conditions

• Functional assays with CDC48 mutants:

  • Express catalytically inactive CDC48 mutants with impaired ATPase domains

  • Use CDC48 antibodies to confirm expression levels

  • Assess failure to rescue phenotypes as demonstration of ATPase-dependent functions

These approaches leverage CDC48 antibodies to elucidate the molecular mechanisms by which CDC48 mediates the extraction and delivery of ERAD substrates to the proteasome for degradation .

How can researchers study public clonotypes of antibodies related to CDC48?

Although not directly focused on CDC48, the methodological approaches used to study public clonotypes (genetically similar antibodies produced by unrelated individuals) can be applied to understand conserved epitopes in CDC48:

• Repertoire analysis:

  • Analyze antibody responses to CDC48 across different individuals

  • Identify convergent antibody sequences that recognize the same CDC48 epitopes

  • Use these insights to identify functionally important and conserved regions of CDC48

• Structural basis of recognition:

  • Study the structural interface between public clonotype antibodies and CDC48

  • Determine whether naive B cell receptors are preconfigured for CDC48 binding

  • Map epitopes that commonly induce antibodies in diverse populations

• Comparative analysis:

  • Compare public clonotypes recognizing different domains of CDC48 (e.g., S1 vs. S2 domains)

  • Identify epitopes that may be more conserved across different strains or species

These approaches could reveal fundamental insights about immunologically significant regions of CDC48 and potentially identify conserved epitopes that could serve as therapeutic targets .

What are common challenges in CDC48 antibody applications and how can they be addressed?

Researchers frequently encounter these challenges when working with CDC48 antibodies:

• Non-specific binding:

  • Increase blocking time and concentration (5% BSA or normal serum)

  • Use higher antibody dilutions (e.g., 1:128,000 for ELISA applications)

  • Include peptide competition controls to verify specificity

  • Pre-adsorb secondary antibodies against tissue lysates

• Inconsistent signal intensity:

  • Standardize protein loading (verify with housekeeping protein controls)

  • Maintain consistent incubation times and temperatures

  • Store antibodies according to manufacturer recommendations (-20°C)

  • Avoid repeated freeze/thaw cycles of antibody aliquots

• Poor signal-to-noise ratio in immunohistochemistry:

  • Optimize fixation protocols (overfixation can mask epitopes)

  • Try different antigen retrieval methods

  • Use thin tissue sections (5-10 μm) as in zebrafish studies

  • Employ fluorophore-conjugated secondary antibodies for better detection

• Variable knockdown efficiency:

  • Titrate morpholino concentrations (1.6-8.0 ng range)

  • Verify knockdown efficiency by Western blot (expect 12-30% of normal CDC48 levels)

  • Include multiple control groups (non-injected and control-MO injected)

Addressing these challenges ensures more reliable and reproducible results when studying CDC48 function and interactions .

How should researchers interpret changes in CDC48 localization during cellular stress?

Changes in CDC48 subcellular distribution require careful interpretation:

• Baseline localization assessment:

  • Under normal conditions, CDC48 localizes to the nucleus, ER, and cytoplasm

  • Establish clear baseline distribution patterns before inducing stress

  • Use z-stack confocal imaging to capture full three-dimensional distribution

• Stress-induced relocalization analysis:

  • Monitor temporal changes in CDC48 distribution following stress induction

  • Quantify relative changes in nuclear vs. cytoplasmic vs. ER localization

  • Correlate with markers of ER stress (e.g., BiP/GRP78) or proteotoxic stress

• Co-localization with functional partners:

  • Assess changes in CDC48 co-localization with cofactors (p47, Ufd1-Npl4)

  • Determine whether stress alters complex formation in specific compartments

  • Correlate with functional outcomes (e.g., accumulation of polyubiquitinated proteins)

• Intervention studies:

  • Test whether overexpression of wild-type CDC48 (but not ATPase-deficient mutants) can reverse stress-induced mislocalization

  • Use pharmacological modulators of ER stress to determine causality

These analytical approaches help determine whether CDC48 relocalization is a cause or consequence of cellular dysfunction in various stress conditions .

What considerations are important when analyzing CDC48 in neurodevelopmental studies?

When investigating CDC48's role in neurodevelopment, researchers should consider:

• Temporal dynamics:

  • Analyze CDC48 expression and localization across developmental timepoints

  • In zebrafish studies, 48 hours post-fertilization (hpf) represents a critical timepoint

  • Track morphological and functional changes in neuronal tissues concurrently

• Region-specific effects:

  • Focus analysis on neuroanatomical regions where CDC48 function is critical:

    • Inner plexiform layer

    • Ganglion cell layer

    • Diencephalon

    • Mesencephalon

  • Compare regions differentially affected by CDC48 deficiency

• Integrated phenotypic assessment:

  • Correlate biochemical findings (CDC48 levels, polyubiquitinated protein accumulation) with:

    • Structural phenotypes (defects in neuronal outgrowth)

    • Functional outcomes (motor deficits, survival)

  • Use microscopic analysis of thin tissue sections (5-10 μm) for detailed morphological evaluation

• Rescue experiment design:

  • Include both wild-type CDC48 and ATPase-deficient mutants

  • Establish dose-response relationships for rescue efficacy

  • Determine minimal CDC48 levels required for normal neurodevelopment

These considerations provide a comprehensive framework for understanding CDC48's essential role in neurodevelopment and the consequences of its dysfunction .

How might advanced imaging techniques enhance CDC48 antibody-based research?

Emerging imaging technologies can significantly expand CDC48 research capabilities:

• Super-resolution microscopy:

  • Overcome diffraction limit to visualize CDC48 distribution at nanometer resolution

  • Track CDC48 hexamer assembly and disassembly in living cells

  • Map CDC48 distribution relative to ubiquitinated substrates with unprecedented precision

• Proximity ligation assays:

  • Detect CDC48 interactions with specific partners in situ

  • Quantify changes in interaction networks during development or disease progression

  • Identify cell type-specific CDC48 complexes in heterogeneous tissues

• Live-cell FRET imaging:

  • Monitor CDC48 conformational changes associated with ATP binding and hydrolysis

  • Measure real-time changes in CDC48 activity under various cellular conditions

  • Detect transient interactions between CDC48 and substrate proteins

• Correlative light and electron microscopy:

  • Combine immunofluorescence localization of CDC48 with ultrastructural context

  • Visualize CDC48 association with specific cellular structures at nanometer resolution

  • Bridge functional and structural studies of CDC48-dependent processes

These advanced imaging approaches will provide unprecedented insights into CDC48's dynamic behavior in living systems .

What potential therapeutic applications might emerge from CDC48 antibody research?

CDC48 antibody research may lead to several therapeutic developments:

• Neuroprotective strategies:

  • Identify compounds that enhance CDC48 function or expression

  • Develop therapies that stabilize CDC48-cofactor interactions

  • Target specific CDC48-dependent degradation pathways implicated in neurodegeneration

• Diagnostic applications:

  • Develop antibody-based assays to detect altered CDC48 function in patient samples

  • Create imaging probes to visualize CDC48 activity in living tissues

  • Establish biomarkers based on CDC48 complex formation or localization

• Precision medicine approaches:

  • Stratify patients based on CDC48 pathway dysfunction profiles

  • Tailor therapeutic interventions to specific CDC48-related defects

  • Monitor treatment efficacy using CDC48 functional readouts

• Gene therapy strategies:

  • Develop targeted delivery of functional CDC48 to affected tissues

  • Create modified CDC48 variants with enhanced neuroprotective properties

  • Design synthetic CDC48 circuits for controlled activity in specific cellular contexts

The essential role of CDC48 in neurodevelopment, combined with the lethal consequences of its dysfunction, highlights its potential as a therapeutic target for neurodegenerative conditions .