CCT8 Antibody

What is CCT8 and why is it important in cellular function?

CCT8 (also known as C21orf112, CCTQ, KIAA0002, CCT-theta, TCP-1-theta, and NY-REN-15) is a crucial subunit of the chaperonin containing t-complex polypeptide 1 (CCT) complex. It belongs to the TCP-1 chaperonin family and functions as a molecular chaperone that assists in protein folding through ATP hydrolysis . As part of the BBS/CCT complex, CCT8 plays a significant role in the assembly of BBSome, a complex involved in ciliogenesis that regulates vesicle transport to cilia . It has been specifically implicated in the folding of critical cytoskeletal proteins including actin and tubulin in vitro .

What types of CCT8 antibodies are available for research applications?

Multiple types of CCT8 antibodies are available for research applications:

These antibodies target different epitopes of CCT8 and have been validated for various experimental applications, making them suitable for different research objectives .

How should I optimize CCT8 antibody concentration for Western blot experiments?

For Western blot applications, recommended dilution ranges vary by antibody:

• Initial titration: Begin with a mid-range dilution (e.g., 1:1000 for most CCT8 antibodies) to establish baseline signal strength.

• Optimization steps:

  • For polyclonal antibodies like 12263-1-AP, start with 1:5000 dilution and adjust as needed

  • For monoclonal antibodies like 67539-1-Ig, a higher dilution range (1:5000-1:50000) may be appropriate

• Cell type considerations: Positive Western blot signals have been consistently detected in multiple cell lines including HEK-293, NIH/3T3, A549, HeLa, Jurkat, K-562, and HSC-T6 cells . Choose an appropriate positive control based on your experimental system.

• Detection method adjustment: For chemiluminescence detection, lower antibody concentrations (higher dilutions) often produce cleaner results with reduced background.

What are the recommended protocols for detecting CCT8 by immunohistochemistry?

For optimal immunohistochemistry detection of CCT8:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded tissues sectioned at 4-6 μm thickness

  • Human liver cancer tissue has been validated as a positive control

• Antigen retrieval:

  • Recommended: TE buffer at pH 9.0

  • Alternative: Citrate buffer at pH 6.0

• Antibody dilution:

  • For monoclonal antibodies like 67539-1-Ig, use 1:1000-1:4000 dilution

• Quantification method:

  • For scoring CCT8 expression in tissue samples, count positively stained cells in high-magnification fields

  • Calculate the integrated optical density (IOD) score by multiplying the intensity score (0-3) by the percentage of immunoreactive cells

  • For comparative analyses, calculate the average optical density (AOD) using the formula: AOD = IOD/Area

How can CCT8 antibodies be utilized to investigate its role in cancer progression?

CCT8 has been identified as a potential prognostic marker in several cancers, particularly lung adenocarcinoma (LUAD). Researchers can utilize CCT8 antibodies to:

• Evaluate expression correlation with patient outcomes:

  • Perform immunohistochemistry on cancer tissue microarrays

  • Calculate AOD scores for CCT8 expression

  • Use the median value of AOD as a cut-off to stratify patients into high and low expression groups

  • Conduct Kaplan-Meier survival analysis to determine the relationship between CCT8 expression and patient survival

• Investigate mechanistic pathways:

  • Perform co-immunoprecipitation assays to identify CCT8 binding partners in cancer cells (e.g., AKT)

  • Use CCT8 antibodies in combination with phospho-specific antibodies to assess activation of signaling pathways

  • In lung cancer models, CCT8 has been shown to interact with and activate AKT, facilitating cell migration and tumor metastasis

• Monitor therapy response:

  • Use CCT8 antibodies to track expression changes during treatment regimens

  • Combine with other markers to develop predictive signatures for therapy response

What approaches can be used to study CCT8's role in protein folding and complex assembly?

To investigate CCT8's chaperonin function:

• Co-immunoprecipitation with ATP release assays:

  • Immunoprecipitate CCT8 with its binding partners (e.g., mLST8, Raptor)

  • Add 5 mM ATP and measure the time-dependent release of substrate proteins

  • Analyze the release kinetics (e.g., half-life of release)

  • CCT8 substrates like mLST8 and Raptor have shown ATP-dependent release from CCT with half-lives of approximately 40 and 37 minutes, respectively

• Cryo-electron microscopy of CCT8-substrate complexes:

  • Isolate CCT8-bound intermediates directly from cells using tandem affinity chromatography

  • Prepare samples for cryo-EM imaging

  • Perform 2D classification and 3D reconstruction to analyze the structural details of the complex

  • This approach has been successfully used to characterize the mLST8-CCT assembly intermediate at 4.0 Å resolution

• Depletion studies to assess functional consequences:

  • Use siRNA to deplete CCT8 in cellular models

  • Assess the impact on target protein folding, complex assembly, and downstream signaling

  • In mTOR signaling studies, CCT8 depletion decreased mLST8 incorporation into mTORC2 by approximately 50%

How can I verify CCT8 antibody specificity and minimize false-positive results?

To ensure antibody specificity:

• Validation controls:

  • Include a positive control from validated cell lines (e.g., Jurkat, MCF-7, HEK-293)

  • Include a negative control by CCT8 knockdown using siRNA

  • For Western blots, verify the expected molecular weight (60 kDa)

• Cross-reactivity testing:

  • Test reactivity against related chaperonin family members

  • Verify species cross-reactivity if working with non-human models

  • Most CCT8 antibodies show 100% predicted reactivity with human, mouse, rat, cow, dog, horse, guinea pig, and yeast samples, and approximately 86% with zebrafish

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide

  • Compare results with and without peptide blocking

  • Signal elimination by the blocking peptide confirms specificity

What are common pitfalls when using CCT8 antibodies in co-immunoprecipitation experiments?

When performing co-immunoprecipitation with CCT8 antibodies:

• Buffer composition considerations:

  • Nucleotide sensitivity: CCT8 interactions with substrates like mLST8 and Raptor are ATP-dependent

  • Including or excluding ATP in buffers can significantly impact results

  • ATP treatment causes time-dependent dissociation of CCT8-substrate complexes

• Technical challenges:

  • Large complexes like Raptor-CCT may be unstable due to domains that don't interact with CCT

  • Consider isolating specific domains (e.g., Raptor's C-terminal β-propeller domain) for more stable interactions

  • For transient interactions, consider crosslinking approaches

• Interpretation considerations:

  • CCT8 functions as part of the larger CCT complex; interactions may involve other subunits

  • Validation with reciprocal immunoprecipitation is essential (pull down with target protein antibody and probe for CCT8, and vice versa)

How can CCT8 antibodies be applied to study its role in viral infection mechanisms?

Recent research has identified CCT8 as a host factor interacting with viral proteins:

• Viral-host protein interaction studies:

  • Use CCT8 antibodies for co-immunoprecipitation with viral proteins

  • Specifically, CCT8 has been shown to interact with the PB2 protein of H9N2 avian influenza virus

  • Perform pulldown assays followed by Western blot or mass spectrometry to identify interaction partners

• Expression dynamics during infection:

  • Monitor CCT8 expression changes during viral infection

  • H9N2 subtype AIV infection induces upregulation of endogenous CCT8 at both mRNA and protein levels

  • Use Western blot with CCT8 antibodies at different time points post-infection

• Functional impact assessment:

  • Combine CCT8 knockdown or overexpression with antibody-based detection of viral proteins

  • Knockdown of CCT8 decreases expression of viral PB2 and NS1 proteins, reducing progeny virus production

  • Conversely, CCT8 overexpression enhances viral protein expression and virus production

What role does CCT8 play in immune cell function, and how can antibodies help elucidate these mechanisms?

CCT8 has been identified as essential for T cell development and function:

• T cell development studies:

  • Use flow cytometry with CCT8 antibodies to assess expression in different T cell subpopulations

  • In CCT8-deficient mice, naive T cell numbers are drastically reduced while memory T cell frequencies are increased

• Proteostasis assessment:

  • Analyze protein expression changes using Western blot with CCT8 antibodies

  • CCT8 deficiency alters the expression of proteins beyond CCT components and tubulin

  • At least six different patterns of protein changes have been detected when comparing mutant and wild-type T cells

• Polarization and functional studies:

  • Use CCT8 antibodies alongside cytokine-specific antibodies to assess T cell polarization

  • CCT8-deficient T cells show defects in Th2 polarization, with paradoxically increased IFN-γ expression

  • Under Treg-inducing conditions, CCT8-deficient cells show reduced FoxP3 expression and unexpected IFN-γ production

How do monoclonal and polyclonal CCT8 antibodies compare in different applications?

For optimal results:

• Choose polyclonal antibodies for applications requiring high sensitivity

• Select monoclonal antibodies for applications requiring high specificity and reproducibility

• Consider using both types complementarily to validate findings

What criteria should guide the selection of CCT8 antibodies for specific research applications?

When selecting a CCT8 antibody, consider:

• Epitope and domain targeting:

  • C-terminal antibodies: Best for detecting full-length CCT8

  • Internal region antibodies: May detect specific functional domains

  • Assess whether the epitope is accessible in your experimental conditions

• Validation data availability:

  • Check for validation in your specific application

  • Review published literature citing the antibody

  • Evaluate the quality of validation data provided by manufacturers

• Species compatibility:

  • Most CCT8 antibodies react with human, mouse, and rat samples

  • For other species, check predicted reactivity percentages

  • Zebrafish studies may require specific antibodies with confirmed reactivity (approximately 86%)

• Application-specific considerations:

  • For co-IP: Choose antibodies validated for immunoprecipitation

  • For IHC: Select antibodies with demonstrated specificity in fixed tissues

  • For multiplexing: Consider host species to avoid cross-reactivity with other primary antibodies