CCT4 Antibody

What is CCT4 and why is it important in biological research?

CCT4 (Chaperonin Containing TCP1 Subunit 4), also known as T-complex protein 1 subunit delta, is a component of the chaperonin-containing T-complex (TRiC), a molecular chaperone complex that assists in protein folding upon ATP hydrolysis. CCT4 has significant importance in cellular biology as it contributes to the folding of various proteins, particularly actin and tubulin. As part of the TRiC complex, it plays critical roles in regulating telomere maintenance and the assembly of BBSome, a complex involved in ciliogenesis that regulates vesicle transport to cilia . Understanding CCT4 function is essential for research into protein folding mechanisms, cellular stress responses, and various pathological conditions including cancer. Recent studies have highlighted CCT4's potential role as a biomarker in hepatocellular carcinoma and other diseases .

What are the typical applications for CCT4 antibodies in research?

CCT4 antibodies are employed across multiple research applications with varying optimization requirements:

When selecting a CCT4 antibody for your research, consider the specific application requirements and choose antibodies validated for your intended application and species of interest .

How can I validate the specificity of a CCT4 antibody?

Validating antibody specificity is crucial for generating reliable research data. For CCT4 antibodies, implement the following validation strategies:

• Positive and negative controls: Use cell lines with known CCT4 expression levels. Several antibodies have been validated in cell lines such as HeLa, MCF-7, HEK-293, and HepG2 .

• Knockdown/knockout validation: Utilize CCT4 siRNA or CRISPR-Cas9 approaches to create CCT4-depleted cells as negative controls. Published studies have employed this approach to confirm antibody specificity .

• Multiple antibody approach: Use different antibodies targeting distinct epitopes of CCT4 to confirm consistent detection patterns.

• Western blot analysis: Confirm the detected band corresponds to the expected molecular weight (55-58 kDa) for CCT4 .

• Peptide competition assay: Pre-incubate the antibody with a blocking peptide (such as the immunogen peptide) to demonstrate signal specificity .

• Cross-reactivity testing: Test the antibody against related proteins, particularly other CCT family members, to ensure specificity within this closely related protein family.

Remember that antibody validation should ideally include both positive results (detection in samples known to express CCT4) and negative controls to conclusively demonstrate specificity .

How does CCT4 expression vary across different tissue types and pathological conditions?

CCT4 expression patterns show distinct variations across tissues and in disease states:

Normal tissues:
CCT4 is expressed in various tissues, with particularly strong expression in tissues with high protein folding demands. The highest expression levels have been observed in brain tissues, particularly in neuronal cells .

Cancer tissues:
Significant upregulation of CCT4 expression has been documented in multiple cancer types, with hepatocellular carcinoma (HCC) being particularly well-studied:

When investigating CCT4 in pathological conditions, it's recommended to use a panel of antibodies and multiple detection methods to accurately characterize expression changes. The observed correlation between CCT4 upregulation and clinical parameters suggests it may serve as a potential prognostic biomarker and therapeutic target, particularly in HCC .

What are the optimal experimental approaches for studying CCT4's interactions within the TRiC complex?

Studying CCT4's interactions within the TRiC complex requires specialized techniques that preserve native protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use CCT4 antibodies to pull down the protein along with its binding partners

  • Western blot for other TRiC components (CCT1-8) to confirm interactions

  • Gentle lysis conditions (non-ionic detergents like NP-40) help preserve complex integrity

• Proximity ligation assay (PLA):

  • Enables visualization of protein-protein interactions in situ

  • Requires primary antibodies from different host species for CCT4 and its interacting partners

  • Can provide spatial information about interactions within subcellular compartments

• Cross-linking mass spectrometry:

  • Chemical cross-linking stabilizes transient interactions

  • MS analysis identifies cross-linked peptides, providing structural information

  • Particularly valuable for understanding the quaternary structure of the TRiC complex

• Bioluminescence resonance energy transfer (BRET)/Fluorescence resonance energy transfer (FRET):

  • Tag CCT4 and potential binding partners with appropriate donor/acceptor pairs

  • Monitor real-time interactions in live cells

  • Useful for studying dynamics of complex assembly/disassembly

When investigating CCT4's role in telomere maintenance, consider chromatin immunoprecipitation (ChIP) experiments to assess potential associations with telomeric regions. For BBSome assembly studies, combine co-IP with sucrose gradient fractionation to isolate complexes at different assembly stages .

How can I design experiments to investigate CCT4's role in cellular stress response and proteostasis?

Investigating CCT4's role in stress response and proteostasis requires a multifaceted experimental approach:

• Stress induction protocols:

  • Heat shock: Expose cells to elevated temperatures (42-45°C) for 30-60 minutes

  • Oxidative stress: Treat with hydrogen peroxide (100-500 μM) or paraquat

  • Proteasome inhibition: Apply MG132 (5-10 μM) for 4-24 hours

  • ER stress: Thapsigargin (1-5 μM) or tunicamycin treatment

  • Monitor CCT4 expression, localization, and activity changes under each condition

• CCT4 modulation techniques:

  • CRISPR-Cas9 knockout: For complete elimination of CCT4

  • siRNA knockdown: For transient reduction (typically 70-90%)

  • Overexpression systems: To assess gain-of-function effects

  • Compare effects on proteostasis markers across these conditions

• Proteostasis assessment:

  • Protein aggregation assays: Filter trap assay, detergent insolubility fractionation

  • Protein folding reporters: Luciferase refolding assay, GFP-fusion protein folding

  • Proteasome activity assays: Fluorogenic substrate cleavage

  • Autophagy flux measurements: LC3-II/LC3-I ratio, p62 degradation

• Client protein analysis:

  • Identify CCT4-dependent substrates using immunoprecipitation followed by mass spectrometry

  • Monitor folding kinetics of known clients (e.g., actin, tubulin) using pulse-chase experiments

  • Assess effects of CCT4 depletion on client protein solubility and function

When analyzing results, consider that the TRiC complex functions as a cohesive unit, so changes in CCT4 may affect the entire complex's functionality. Include additional chaperone markers (HSP70, HSP90) to determine whether observed effects are specific to TRiC or reflect broader changes in the proteostasis network .

What is currently known about the relationship between CCT4 expression and cancer progression?

The relationship between CCT4 expression and cancer progression has been extensively studied, particularly in hepatocellular carcinoma:

For researchers studying CCT4 in cancer, it's advisable to employ multiparameter analysis combining expression levels with genetic alterations and correlate these with clinical parameters. The evidence suggests CCT4 could serve as both a prognostic biomarker and potential therapeutic target, particularly in HCC, though further validation studies are needed .

What are the optimal sample preparation protocols for CCT4 antibody applications?

Sample preparation is critical for successful CCT4 detection across different applications:

For Western Blotting:

• Cell lysis: Use RIPA buffer or NP-40 based lysis buffers with protease inhibitors

• Protein estimation: BCA or Bradford assay to ensure equal loading

• Sample denaturation: Heat at 95°C for 5 minutes in Laemmli buffer with DTT or β-mercaptoethanol

• Recommended loading: 20-50 μg of total protein per lane

• Separation: 10-12% SDS-PAGE gels provide optimal resolution for CCT4 (55-58 kDa)

For Immunohistochemistry:

• Fixation: 10% neutral buffered formalin for 24-48 hours

• Processing: Standard paraffin embedding

• Sectioning: 4-5 μm sections

• Antigen retrieval: Most effective with TE buffer pH 9.0 (alternative: citrate buffer pH 6.0)

• Blocking: 5-10% normal serum (matching secondary antibody host) with 1% BSA

For Immunofluorescence:

• Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilization: 0.1-0.5% Triton X-100 for 5-10 minutes

• Blocking: 5% BSA or 10% normal serum for 30-60 minutes

• Antibody incubation: Overnight at 4°C or 1-2 hours at room temperature

For Flow Cytometry:

• Fixation: 2-4% paraformaldehyde

• Permeabilization: Critical for intracellular CCT4 detection using 0.1% saponin or commercial permeabilization buffers

• Blocking: 5% normal serum or 2% BSA

• Single cell preparation: Gentle cell dissociation to maintain epitope integrity

Always validate these protocols for your specific cell type or tissue and adjust parameters as needed for optimal results.

What are the common troubleshooting issues with CCT4 antibodies and how can they be addressed?

Researchers commonly encounter several issues when working with CCT4 antibodies. Here are solutions to the most frequent problems:

Weak or No Signal in Western Blot:

• Increase protein loading (50-100 μg)

• Optimize primary antibody concentration (try serial dilutions)

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

• Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

• Check transfer efficiency with reversible protein stains (Ponceau S)

High Background in Immunostaining:

• Increase blocking time (2 hours) and concentration (5-10% serum)

• Add 0.1-0.3% Triton X-100 to antibody diluent

• Include 0.05-0.1% Tween-20 in wash buffers

• Use more stringent washing (increase number and duration of washes)

• Try alternative blocking agents (2% BSA, 5% milk, commercial blockers)

Multiple Bands in Western Blot:

• Use fresh sample preparation with protease inhibitors

• Include phosphatase inhibitors if studying post-translational modifications

• Increase gel percentage for better resolution

• Verify with CCT4 knockout/knockdown controls

• Consider that isoforms (55 kDa and 58 kDa) may be detected

Cross-reactivity Issues:

• Use affinity-purified antibodies

• Validate with peptide competition assays

• Employ antibodies targeting non-conserved regions of CCT4

• Pre-adsorb antibody with related proteins

• Include knockout/knockdown controls to verify specificity

Variable Results Between Experiments:

• Standardize sample preparation protocols

• Include internal loading controls (β-actin, GAPDH)

• Prepare larger batches of antibody dilutions

• Document lot numbers and maintain consistent reagent sources

• Employ positive controls (cell lines with known CCT4 expression)

When troubleshooting persistent issues, consider testing multiple antibodies against different epitopes of CCT4 to determine if the problem is antibody-specific or related to your experimental system.

How can quantitative analysis of CCT4 expression be accurately performed?

Accurate quantitative analysis of CCT4 expression requires rigorous methodology and appropriate controls:

Western Blot Quantification:

• Ensure linear range of detection:

  • Load a concentration gradient of samples

  • Verify signal is within linear range of detection system

• Normalization strategies:

  • Use housekeeping proteins (GAPDH, β-actin) for loading control

  • Consider total protein normalization (Ponceau S, SYPRO Ruby) for more accurate results

• Image acquisition:

  • Use cooled CCD camera systems rather than film

  • Avoid signal saturation

• Analysis software:

  • ImageJ/FIJI (free), Image Lab, or similar densitometry software

  • Define consistent measurement parameters across all samples

Immunohistochemistry Quantification:

• Scoring systems:

  • H-score (intensity × percentage of positive cells)

  • Allred score (combines intensity and proportion)

  • Digital image analysis with color deconvolution

• Observer variability reduction:

  • Blinded analysis by multiple observers

  • Use of automated scanning and analysis platforms

• Controls for normalization:

  • Include reference tissues with known expression levels on same slide

  • Use serial sections for technical replicates

Flow Cytometry Quantification:

• Standardization:

  • Use calibration beads to standardize fluorescence intensity

  • Establish defined gates based on negative controls

• Analysis parameters:

  • Mean/median fluorescence intensity (MFI)

  • Percentage of positive cells above threshold

• Controls:

  • FMO (fluorescence minus one) controls

  • Isotype controls matched to primary antibody

RT-qPCR for mRNA Quantification:

• Reference gene selection:

  • Use multiple reference genes validated for your experimental system

  • Verify reference gene stability across conditions

• Analysis methods:

  • ΔΔCt method with appropriate normalization

  • Standard curve method for absolute quantification

When comparing CCT4 expression across different conditions or disease states, always process and analyze all samples simultaneously to minimize batch effects. For clinical correlations, employ multivariate statistical analyses to account for confounding variables .

How can CCT4 antibodies be used to investigate its role in cancer biomarker studies?

CCT4 antibodies are valuable tools for investigating its potential as a cancer biomarker, particularly in hepatocellular carcinoma (HCC). Here's a methodological approach for such studies:

• Tissue microarray (TMA) analysis:

  • Create TMAs containing tumor tissues, adjacent normal tissues, and normal controls

  • Perform IHC using optimized CCT4 antibodies (1:20-1:200 dilution)

  • Quantify expression using H-score or digital image analysis

  • Correlate with clinicopathological parameters including:

    • Tumor stage and grade

    • Vascular invasion status

    • Alpha-fetoprotein levels

    • Survival outcomes

• Multiplex immunofluorescence:

  • Co-stain for CCT4 and other cancer markers

  • Assess cellular localization and co-expression patterns

  • Combine with markers of proliferation (Ki-67) or other oncogenic pathways

• Liquid biopsy approaches:

  • Develop assays to detect CCT4 in patient serum or circulating tumor cells

  • Compare with established biomarkers (e.g., AFP for HCC)

  • Evaluate sensitivity and specificity for early detection

• Prognostic correlation studies:

  • Stratify patients based on CCT4 expression levels

  • Perform Kaplan-Meier survival analysis

  • Calculate hazard ratios using Cox regression

  • Compare with established prognostic markers

• Therapeutic response prediction:

  • Assess CCT4 expression before and after treatment

  • Correlate expression levels with response to specific therapies

  • Develop predictive models incorporating CCT4 status

What methods are recommended for studying functional roles of CCT4 in protein folding?

Investigating CCT4's functional roles in protein folding requires specialized techniques spanning biochemical, cellular, and structural approaches:

• In vitro folding assays:

  • Luciferase refolding: Measure refolding of denatured luciferase in the presence/absence of functional CCT4

  • Target protein folding kinetics: Monitor folding of known CCT4 clients (actin, tubulin) using circular dichroism or fluorescence spectroscopy

  • ATP hydrolysis coupling: Correlate ATPase activity with folding efficiency

• Client protein identification:

  • Co-immunoprecipitation with CCT4-specific antibodies followed by mass spectrometry

  • Proximity labeling approaches (BioID, APEX) with CCT4 as the bait protein

  • Differential analysis of protein aggregation following CCT4 depletion

• CCT4 loss-of-function studies:

  • siRNA knockdown: Transient reduction of CCT4 (70-90%)

  • CRISPR-Cas9 knockout: Complete elimination (consider inducible systems as complete loss may be lethal)

  • Dominant-negative mutants: Express ATPase-deficient CCT4 to disrupt function

• Real-time folding visualization:

  • FRET-based folding sensors to monitor conformational changes in client proteins

  • Pulse-chase experiments to track nascent protein folding

  • Live-cell imaging with fluorescently tagged client proteins

• Structure-function studies:

  • Site-directed mutagenesis of key CCT4 residues

  • Cryo-EM analysis of TRiC complex with client proteins in different states

  • Crosslinking mass spectrometry to map interaction interfaces

When designing these experiments, consider that CCT4 functions as part of the larger TRiC complex, so isolated studies of CCT4 may not fully recapitulate its physiological function. Include complementation experiments (rescuing knockdown/knockout with wild-type vs. mutant CCT4) to establish specific functional roles .

How can CCT4 be targeted for therapeutic development and what role do antibodies play in this research?

The emerging role of CCT4 in cancer progression makes it an attractive therapeutic target. Antibodies serve multiple functions in this research area:

• Target validation approaches:

  • Immunohistochemical profiling across cancer types to identify high-expression tumors

  • Correlation of expression with clinical outcomes to establish prognostic significance

  • CCT4 knockdown/knockout studies to confirm dependency in cancer models

• Drug development support:

  • Screening assays: Use CCT4 antibodies in ELISA or AlphaScreen formats to identify small molecule binders

  • Target engagement: Cellular thermal shift assays (CETSA) with CCT4 antibodies to confirm compound binding

  • Mechanism of action studies: Evaluate effects on CCT4 protein interactions using co-IP with CCT4 antibodies

• Antibody-based therapeutics:

  • Intrabodies: Engineered antibody fragments expressed intracellularly to inhibit CCT4 function

  • Antibody-drug conjugates: For cancers with cell-surface expression or during internalization

  • CAR-T targeting: If tumor-specific expression patterns can be established

• Pathway modulation assessment:

  • Monitor PI3K/AKT pathway components following CCT4 modulation

  • Evaluate effects on client protein folding and function

  • Assess combination therapy approaches targeting CCT4 and related pathways

• Predictive biomarker development:

  • Develop standardized IHC protocols to stratify patients for clinical trials

  • Create companion diagnostic assays using validated CCT4 antibodies

  • Monitor treatment response through serial sampling (liquid biopsy approaches)

Research indicates that CCT genetic alterations affect the PI3K/AKT pathway, suggesting potential synergy between CCT4-targeted therapies and existing PI3K/AKT inhibitors. The significant association of CCT4 upregulation with poor prognosis in HCC provides strong rationale for therapeutic development in this indication .

When developing therapeutics targeting CCT4, consider its essential role in normal cell physiology and establish a therapeutic window that allows targeting of cancer cells while minimizing toxicity to normal tissues.

What are the best experimental approaches for studying CCT4's role in specific cellular processes like ciliogenesis?

Investigating CCT4's role in specific processes like ciliogenesis requires specialized experimental approaches:

• Ciliary structure and formation analysis:

  • Primary cilium induction: Serum starvation (24-48 hours) in appropriate cell lines (RPE-1, IMCD3)

  • CCT4 manipulation: siRNA knockdown, CRISPR knockout, or overexpression

  • Immunofluorescence microscopy:

    • Use antibodies against ciliary markers (acetylated tubulin, Arl13b) with CCT4 co-staining

    • Quantify cilia number, length, and morphology

    • Image acquisition with high-resolution confocal or super-resolution microscopy

• BBSome assembly and function:

  • Co-immunoprecipitation: Use CCT4 antibodies to pull down complex and probe for BBSome components

  • Sucrose gradient fractionation: Analyze BBSome assembly intermediates with/without CCT4

  • FRAP (Fluorescence Recovery After Photobleaching): Study dynamic transport of BBSome components in the presence/absence of CCT4

  • Rescue experiments: Express wild-type vs. mutant CCT4 in depleted cells

• Live imaging approaches:

  • Fluorescently-tagged CCT4: Monitor localization during ciliogenesis

  • Time-lapse microscopy: Track ciliary growth kinetics following CCT4 perturbation

  • Photoactivatable or photoswitchable tags: Monitor protein movement and turnover

• Biochemical interaction studies:

  • In vitro binding assays with purified components

  • Proximity labeling (BioID, APEX) with CCT4 as bait in ciliated cells

  • Crosslinking mass spectrometry to identify interaction interfaces

• Functional readouts:

  • Hedgehog signaling assays: Gli1 transcriptional activity using luciferase reporters

  • Ciliary protein trafficking: Monitor Smoothened localization to cilia following Hedgehog pathway activation

  • Cell migration assays: Wound healing or transwell migration to assess functional consequences

When designing these experiments, consider the timeframe of ciliogenesis (typically 24-48 hours post-serum starvation) and ensure CCT4 manipulation is maintained throughout this period. For transient knockdown approaches, confirm protein depletion at the endpoint of your experiment, as protein turnover rates may affect interpretation of results .

What are the emerging roles of CCT4 in disease pathogenesis beyond cancer?

While CCT4's role in cancer has been extensively studied, emerging research suggests broader implications in various disease processes:

• Neurodegenerative disorders:

  • Protein misfolding is central to neurodegenerative diseases

  • CCT4 may influence α-synuclein and tau protein folding

  • Brain tissues show high CCT4 expression, suggesting neuronal importance

  • Research methods:

    • Animal models expressing disease-associated protein aggregates

    • CCT4 modulation in neuronal cell cultures

    • Co-localization studies with disease-specific protein aggregates

• Cardiovascular disease:

  • Proteostasis disruption contributes to cardiomyopathies

  • CCT4's role in cytoskeletal protein folding may affect cardiac function

  • Experimental approaches:

    • Tissue-specific CCT4 knockout in cardiac models

    • Pressure overload studies with CCT4 modulation

    • Analysis of CCT4 expression in heart failure samples

• Infectious diseases:

  • Some pathogens may hijack host chaperone systems

  • CCT4 could be involved in viral protein folding or assembly

  • Investigation methods:

    • Infection models with CCT4 knockdown

    • Interaction studies between viral proteins and CCT4

    • Small molecule modulators of CCT4 as potential antivirals

• Metabolic disorders:

  • Protein quality control plays a role in obesity and diabetes

  • CCT4 may influence insulin signaling pathway components

  • Approaches:

    • Analysis in metabolic tissues (liver, adipose, pancreas)

    • Diet-induced obesity models with CCT4 modulation

    • Glucose tolerance testing following CCT4 manipulation

Future research should focus on tissue-specific functions of CCT4 and developing conditional knockout models to address potential embryonic lethality of complete CCT4 loss. The development of specific small molecule modulators of CCT4 function would provide valuable tools for investigating its role in these various disease contexts.

What technological advances are improving CCT4 antibody development and applications?

Recent technological advances are significantly enhancing CCT4 antibody quality, specificity, and application range:

• Recombinant antibody technology:

  • Single B cell cloning from immunized animals

  • Phage display libraries for epitope-specific selection

  • Advantages:

    • Reduced batch-to-batch variability

    • Renewable source without animal immunization

    • Potential for engineering enhanced properties

• Nanobody and single-domain antibody platforms:

  • Smaller antigen-binding fragments with improved tissue penetration

  • Applications:

    • Super-resolution microscopy with reduced linkage error

    • Intracellular expression as functional inhibitors ("intrabodies")

    • Improved accessibility to conformational epitopes

• Multiplexed antibody validation:

  • CRISPR knockout cell lines as gold-standard controls

  • Automated high-throughput validation pipelines

  • Orthogonal validation across multiple applications

  • Standardized reporting of validation data

• Advanced imaging applications:

  • Expansion microscopy compatible antibodies

  • DNA-PAINT and other super-resolution techniques

  • Clearing-compatible antibodies for 3D tissue imaging

  • Applications to visualize CCT4 in cellular nanocompartments

• Antibody conjugates and modifications:

  • Site-specific conjugation strategies

  • Novel fluorophores with improved properties

  • Bifunctional antibodies for proximity detection

  • Mass cytometry (CyTOF) compatible metal conjugates

These technological advances enable more precise investigation of CCT4's subcellular localization, interaction partners, and functional roles across different physiological and pathological contexts. Researchers should stay informed about validation standards and emerging technologies to select the most appropriate antibodies for their specific applications.

How can systems biology approaches incorporate CCT4 antibody data to understand broader cellular networks?

Systems biology offers powerful frameworks to contextualize CCT4 within broader cellular networks:

• Multi-omics data integration:

  • Combine antibody-based proteomics with:

    • Transcriptomics (RNA-seq) to correlate mRNA and protein levels

    • Phosphoproteomics to map post-translational modifications

    • Interactomics (IP-MS) to define protein-protein interactions

  • Integration methods:

    • Network analysis tools (Cytoscape, STRING)

    • Correlation analysis across data types

    • Machine learning approaches for pattern recognition

• Pathway analysis and modeling:

  • Map CCT4 interactions to known signaling pathways

  • Identify enriched pathways following CCT4 perturbation

  • Create mathematical models of chaperone networks

  • Simulate effects of CCT4 modulation on proteostasis

• Single-cell multi-parameter analysis:

  • Single-cell proteomics with CCT4 antibodies

  • Mass cytometry (CyTOF) for high-dimensional analysis

  • Multiplexed imaging (CODEX, MIBI) for spatial context

  • Reveals cell-to-cell variability in CCT4 expression and function

• Perturbation biology approaches:

  • CRISPR screens combined with CCT4 antibody readouts

  • Small molecule library screening

  • Genetic interaction mapping (synthetic lethality)

  • Identifies context-dependent functions and vulnerabilities

• Clinical data correlation:

  • Integrate CCT4 expression data with:

    • Patient outcomes and survival

    • Treatment responses

    • Genetic backgrounds

  • Build predictive models for personalized medicine approaches

These approaches can reveal how CCT4 functions within the broader chaperone network and proteostasis system. For example, research has already identified connections between CCT4 genetic alterations and the PI3K/AKT pathway in HCC, demonstrating how systemic approaches can uncover unexpected functional relationships .

When implementing these approaches, standardization of antibody-based detection methods is crucial for generating comparable datasets across different studies and experimental platforms.