CCT3 Antibody
Description
Introduction to CCT3 Protein
CCT3, also known as T-complex protein 1 subunit gamma, is encoded by the CCT3 gene located on human chromosome 1. As a fundamental component of the TRiC (TCP-1 Ring Complex) chaperonin complex, CCT3 plays an essential role in protein folding mechanisms within cells . The TRiC complex consists of two identical stacked rings, each containing eight distinct protein subunits, forming a barrel-shaped structure with a central cavity where protein folding occurs in an ATP-dependent manner . This complex is particularly important for folding structural proteins such as actin and tubulin, which are critical for maintaining cellular architecture and function .
The CCT3 protein comprises 545 amino acids with a predicted molecular weight of approximately 60-61 kDa . Its structure incorporates domains necessary for ATP binding, substrate recognition, and interaction with other TRiC complex components. Multiple alternative splice variants of CCT3 have been characterized, potentially contributing to diverse functional roles in different cellular contexts .
General Properties
CCT3 antibodies are immunoglobulins specifically designed to recognize and bind to CCT3 protein with high affinity and specificity. These antibodies serve as invaluable tools for detecting, quantifying, and visualizing CCT3 protein in various experimental applications. Commercial CCT3 antibodies are available in different formats, including polyclonal and monoclonal variants, each with specific advantages depending on the research application.
Alternative Names and Identifiers
The target protein CCT3 is known by several alternative names in scientific literature, including CCT gamma, CCT-gamma, CCTG, TCP-1-gamma, hTRiC5, PIG48, and TCPG_HUMAN . This diversity of nomenclature reflects the historical development of research on this protein and its incorporation into different protein complexes.
Western Blotting
CCT3 antibody is extensively used in Western blotting for detecting and quantifying CCT3 protein in cell and tissue lysates. This technique enables researchers to determine CCT3 expression levels, particularly in comparative studies between normal and pathological states. A standard Western blotting protocol for CCT3 detection typically involves:
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Sample preparation and protein extraction from cells or tissues
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Protein separation by SDS-PAGE
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Transfer to a polyvinylidene difluoride (PVDF) membrane
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Blocking with 5% skimmed milk
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Incubation with primary CCT3 antibody (typically at 1:1000 dilution)
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Detection using HRP-conjugated secondary antibodies (e.g., goat anti-rabbit IgG at 1:5000 dilution)
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Signal development and image acquisition
Western blot analysis has been successfully employed to verify CCT3 knockdown following siRNA transfection, demonstrating the antibody's utility in validating experimental manipulations of CCT3 expression .
Immunohistochemistry
CCT3 antibody is valuable for immunohistochemical (IHC) detection of CCT3 protein in tissue sections, enabling visualization of CCT3 expression patterns within the tissue architecture. This application has been particularly important in cancer research, where differential expression of CCT3 between normal and malignant tissues has been documented .
In lung adenocarcinoma (LUAD) research, immunohistochemical staining using CCT3 antibody has revealed significantly higher staining intensity in LUAD tissues compared to normal lung tissues, providing visual confirmation of CCT3 overexpression in this cancer type .
Immunofluorescence and Immunocytochemistry
CCT3 antibody can be applied in immunofluorescence (IF) and immunocytochemistry (ICC) techniques to visualize the subcellular localization of CCT3 protein within cells. These techniques provide valuable insights into the spatial distribution of CCT3 in relation to other cellular components and structures, enhancing our understanding of its functional roles in different cellular compartments .
CCT3 Overexpression in Lung Adenocarcinoma
CCT3 antibody has been instrumental in establishing the overexpression of CCT3 in lung adenocarcinoma (LUAD) at both mRNA and protein levels. Comprehensive analyses using various databases, including UALCAN, Human Protein Atlas (HPA), and The Cancer Genome Atlas (TCGA), have demonstrated significantly elevated CCT3 expression in LUAD compared to normal lung tissues .
Specifically, the Cancer Proteome Tumor Analysis Consortium (CPTAC) dataset, accessed through the UALCAN portal, revealed significantly higher CCT3 protein expression in LUAD tumor tissues compared to normal lung tissues (P = 2.464e-18, with 111 samples in each group) . This finding was further corroborated by immunohistochemical results from the Human Protein Atlas database, which showed markedly higher CCT3 staining intensity in LUAD tissues relative to normal lung tissues .
CCT3 and Tumor Microenvironment
Research employing CCT3 antibody has contributed to understanding the relationship between CCT3 expression and the tumor immune microenvironment. Single-sample Gene Set Enrichment Analysis (ssGSEA) has revealed significant correlations between CCT3 expression and specific immune cell populations:
Table 2: Correlation Between CCT3 Expression and Immune Cell Infiltration in LUAD
| Immune Cell Type | Correlation Coefficient | P-value |
|---|---|---|
| Th2 cells | +0.442 | <0.01 |
| Mast cells | -0.49 | <0.01 |
| Immature dendritic cells (iDCs) | -0.401 | <0.001 |
These findings suggest that CCT3 may modulate the tumor immune microenvironment, potentially contributing to immune evasion mechanisms in LUAD .
CCT3 as a Prognostic Biomarker
Furthermore, multivariate Cox hazard regression analysis identified CCT3 expression, along with pathological stage, as an independent prognostic factor for LUAD, highlighting its potential clinical utility in risk stratification and treatment planning .
CCT3 Knockdown Studies and Antibody Validation
CCT3 antibody has been crucial in validating CCT3 knockdown experiments, providing direct evidence of successful protein depletion following siRNA-mediated gene silencing. In LUAD research, siRNA-mediated CCT3 knockdown in A549 cells was verified by Western blotting using CCT3 antibody .
The knockdown experiments utilized specific siRNA sequences targeting CCT3:
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siCCT3 #1: 5'-GGG ACC ACA UCA GUA AUU ATT-3'
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siCCT3 #2: 5'-UAA UUA CUG AUG UGG UCC CTT-3'
Compared with control siRNAs:
These experiments demonstrated that CCT3 knockdown substantially inhibited proliferation and promoted apoptosis in A549 lung adenocarcinoma cells, confirming CCT3's oncogenic role in LUAD .
CCT3-Associated Gene Networks
Research employing CCT3 antibody has contributed to mapping CCT3-related gene networks. Analysis using the GeneMANIA database identified a network of 20 CCT3-associated genes, including:
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Other TRiC complex components: CCT2, TCP1, CCT4, CCT7, CCT6A, CCT6B, CCT5, CCT8
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Additional interacting proteins: IGBP1, ARPC1A, PDCL3, MKKS, SPHK1, PFDN1/4/6/2/5, PDCD5, and WDR77
These associated genes are functionally connected to processes involving the assembly and stability of proteins related to cytoskeletal filaments, DNA repair, and protein methylation .
CCT3-Related Signaling Pathways
Gene Set Enrichment Analysis (GSEA) has revealed that high CCT3 expression is associated with enrichment of specific cellular pathways:
Table 3: Pathways Enriched in CCT3-High vs. CCT3-Low Groups
| CCT3 Expression Level | Enriched Pathways |
|---|---|
| CCT3-High | Cell cycle pathway, Protein export pathway, Proteasome pathway, Ribosome pathway |
| CCT3-Low | JAK/STAT pathway, B cell receptor pathway, T cell receptor pathway, Toll-like receptor pathway |
This distinct pathway enrichment pattern suggests that CCT3 may influence cancer progression through modulation of cell cycle, protein homeostasis, and immune-related signaling pathways .
Optimization of Antibody Dilution
Optimal dilution of CCT3 antibody may vary depending on the specific application and experimental conditions. For Western blotting, a 1:1000 dilution is typically recommended , while immunohistochemistry and immunofluorescence applications may require different dilutions. It is advisable to perform titration experiments to determine the optimal antibody concentration for each specific application and sample type.
Cross-Reactivity Considerations
When working with samples from species not directly validated for reactivity with a particular CCT3 antibody, cross-reactivity potential should be considered. The high conservation of CCT3 sequence across species often enables cross-reactivity with CCT3 from multiple species, but this should be empirically validated for critical experiments .
Controls for Antibody Validation
Proper controls are essential for validating CCT3 antibody specificity and performance:
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Positive controls: Samples known to express CCT3 (e.g., A549 cells)
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Negative controls: Samples with CCT3 knockdown or tissues known to express minimal CCT3
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Secondary antibody-only controls: To evaluate non-specific binding of the secondary antibody
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Isotype controls: To assess non-specific binding of the primary antibody
Product Specs
Target Background
- Studies suggest that CCT3 is crucial for the proliferation of hepatocellular carcinoma (HCC) cells, making it a potential therapeutic target for HCC treatment. PMID: 26739059
- Overexpression of CCT3 within the nuclei of cancerous cells is associated with HCC progression. CCT3 may be a target that influences the activation of STAT3 in HCC. PMID: 26229401
- Expression of S100A9 and CCTgamma was significantly elevated in liver tissue of patients with cholangiocarcinoma compared to control subjects. PMID: 23868055
- Among ten HCC cases with amplicon 1q21-q22, significant gene expression levels of JTB, SHC1, CCT3 and COPA were observed in the tumors compared to the paired adjacent non-malignant liver tissues. PMID: 12586295
Q&A
Basic Research: Antibody Selection and Validation
Q1: What criteria should researchers prioritize when selecting CCT3 antibodies for cancer studies?
A1: Prioritize antibodies validated in relevant applications (e.g., IHC, WB, IP) and tissues. Key factors include:
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Epitope specificity: Target regions (e.g., aa 300–350 or 450–C-term) influence detection accuracy .
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Species reactivity: Confirm cross-reactivity with human/mouse samples .
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Experimental validation: Use knockdown controls (siRNA/CRISPR) to confirm signal specificity .
| Antibody Source | Epitope | Applications | Species |
|---|---|---|---|
| Abcam (ab176686) | aa 450–C-term | IP, WB, IHC-P | Human, Mouse |
| Abcam (ab225878) | aa 300–350 | IHC-P, IP, WB | Human, Mouse |
Q2: How can researchers validate antibody specificity for CCT3?
A2: Implement multi-layered validation:
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Western blot controls: Use lysates from CCT3-knockdown cells (siRNA/CRISPR) to confirm loss of signal .
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Immunoprecipitation (IP): Co-IP with TRiC subunits (e.g., CCT4, CCT5) to confirm complex association .
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IHC negative controls: Omit primary antibody or use isotype-matched IgG .
Advanced Research: Mechanistic Insights and Data Integration
Q3: How does CCT3 modulate the tumor immune microenvironment, and what antibody-based methods can study this?
A3: CCT3 suppresses immune infiltration via:
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Negative correlation with immune checkpoint genes (e.g., CD274, PDCD1) .
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Inhibition of cytotoxic T-cell activity and NK cell cytotoxicity .
Experimental approaches:
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Multiplex IHC: Co-stain CCT3 with immune markers (e.g., CD8, PD-1) to map spatial interactions .
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Single-cell sequencing: Use antibodies to profile CCT3 expression in immune subsets (e.g., Tregs, macrophages) .
Q4: What strategies address conflicting data on CCT3’s oncogenic role across cancers?
A4: Resolve discrepancies via:
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Tissue-specific validation: Use cancer-type-specific cell lines (e.g., COAD for colon, LUAD for lung) .
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Pathway analysis: Perform GSEA to link CCT3 expression to cancer-specific processes (e.g., amino acid metabolism in COAD) .
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Proteomics integration: Combine antibody data with mass spectrometry to identify context-dependent interactomes (e.g., ACTN4 in HCC) .
Experimental Design and Optimization
Q5: How to optimize IHC protocols for detecting CCT3 in formalin-fixed paraffin-embedded (FFPE) tissues?
A5: Implement:
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Antigen retrieval: Use high-temperature epitope retrieval (e.g., Tris-EDTA buffer) .
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Signal amplification: Apply tyramide-based systems for low-abundance targets .
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Negative controls: Include non-cancerous tissue sections to assess background .
Q6: What role does CCT3 play in drug resistance, and how to study this with antibodies?
A6: CCT3 promotes resistance via:
Antibody-based assays:
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Co-IP: Detect CCT3-ACTN4 interactions under drug treatment .
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Flow cytometry: Measure TFRC membrane localization post-CCT3 knockdown .
Emerging Frontiers and Technical Challenges
Q7: How to study CCT3’s subcellular localization in cancer cells?
A7: Use:
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Confocal microscopy: Co-stain CCT3 with organelle markers (e.g., ER, Golgi) .
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BioID proximity labeling: Map interactions in specific compartments .
Q8: What challenges arise when using antibodies to study CCT3’s interaction networks?
A8: Key challenges include:
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Cross-reactivity: Ensure antibodies distinguish CCT3 from other TRiC subunits (e.g., CCT4) .
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Dynamic interactions: Use time-course experiments to capture transient complexes (e.g., Sorafenib-induced CCT3-ACTN4 binding) .
Therapeutic and Prognostic Implications
Q9: How does CCT3’s role in amino acid metabolism inform therapeutic strategies?
A9: Targets include:
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CCT3 inhibitors: Disrupt cell cycle (e.g., AT-7519 in NSCLC) .
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Metabolic synergies: Combine CCT3 inhibitors with anti-ferroptosis agents .
Q10: What prognostic value does CCT3 hold in cancer subtypes?
A10: High CCT3 correlates with:
Methodological Integration
Q11: How to integrate CCT3 antibody data with single-cell sequencing?
A11: Apply:
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Spatial transcriptomics: Map CCT3 protein expression to immune cell clusters .
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Multi-omics correlation: Link CCT3 protein levels to scRNA-seq signatures (e.g., YAP1 activation) .
Q12: What computational tools enhance CCT3 antibody study design?
A12: Utilize:
Future Directions
Q13: How might antibodies enable precision targeting of CCT3 in cancers?
A13: Potential approaches:
