CCT7 Antibody, FITC conjugated

What is CCT7 and why is it important in molecular biology research?

CCT7 (chaperonin containing TCP1 subunit 7) is a critical component of the TRiC/CCT chaperonin complex, which consists of two stacked rings, each containing eight different subunits. This complex plays an essential role in the ATP-dependent folding of approximately 10% of newly synthesized proteins, with particular importance for actin and tubulin folding . The significance of CCT7 in research stems from its fundamental role in protein homeostasis, making it relevant to multiple biological processes including cytoskeletal organization, cell cycle progression, and protein quality control mechanisms .

How do FITC-conjugated CCT7 antibodies differ from unconjugated antibodies in experimental applications?

FITC-conjugated CCT7 antibodies offer direct visualization capabilities through fluorescence microscopy without requiring secondary antibodies, streamlining immunofluorescence workflows . This conjugation allows for:

• Direct detection in flow cytometry and immunofluorescence microscopy

• Multiplexing with antibodies carrying different fluorophores

• Reduced background signal compared to two-step detection methods

• Preservation of native protein interactions that might be disrupted by secondary antibody binding

What are the recommended storage conditions for maintaining FITC-conjugated CCT7 antibody integrity?

FITC-conjugated antibodies require specific storage conditions to maintain both antibody functionality and fluorophore activity. All conjugated antibodies should be stored in light-protected vials or covered with a light-protecting material (such as aluminum foil) . While conjugated antibodies remain stable for at least 12 months at 4°C, longer storage (up to 24 months) requires dilution with up to 50% glycerol and storage at -20°C to -80°C . It's important to note that repeated freezing and thawing will compromise both enzyme activity and antibody binding capacity . For optimal performance, aliquoting upon receipt is recommended to minimize freeze-thaw cycles.

What are the optimal fixation and permeabilization methods when using FITC-conjugated CCT7 antibodies for immunofluorescence?

When using FITC-conjugated CCT7 antibodies for immunofluorescence microscopy, the fixation and permeabilization methods significantly impact epitope accessibility and fluorescence intensity. Based on research protocols:

For permeabilization, research protocols indicate optimal results with 0.1% Triton X-100 in 2% bovine serum albumin (BSA) solution for 10 minutes at room temperature, followed by blocking with 2% BSA for 1 hour . This approach maintains CCT7 epitope integrity while allowing sufficient antibody penetration into the cell.

How should colocalization studies with CCT7 and its client proteins be designed and analyzed?

Colocalization studies investigating CCT7 interactions with client proteins require careful experimental design. Research indicates successful protocols involve:

• Sequential antibody application: When using multiple antibodies, apply primary antibodies sequentially with thorough washing steps to prevent cross-reactivity.

• Controls: Include single-stained samples to establish proper compensation and identify potential spectral overlap between fluorophores.

• Image acquisition parameters:

  • Use sequential scanning to prevent bleed-through

  • Match pinhole settings across all channels

  • Optimize laser power to prevent photobleaching of FITC

• Quantitative analysis:

  • Calculate Pearson's correlation coefficient and Mander's overlap coefficient

  • Perform intensity correlation analysis

  • Use object-based colocalization for punctate structures

Research has successfully demonstrated colocalization between CCT7 and viral proteins such as VP2 using this approach . For example, F81 cells co-transfected with pDsRed-CCT7 and pEGFP-VP2 plasmids showed distinct colocalization patterns when visualized using confocal microscopy .

What controls should be included when using FITC-conjugated CCT7 antibodies in flow cytometry experiments?

When performing flow cytometry with FITC-conjugated CCT7 antibodies, including appropriate controls is essential for accurate data interpretation:

For intracellular CCT7 detection, proper permeabilization validation is crucial, as incomplete permeabilization can lead to false negatives in CCT7 detection .

How can FITC-conjugated CCT7 antibodies be used to investigate protein-folding dynamics in live cells?

Investigating protein-folding dynamics in live cells using FITC-conjugated CCT7 antibodies presents technical challenges but offers valuable insights. Advanced approaches include:

• Microinjection of antibodies: Carefully calibrated microinjection of FITC-conjugated CCT7 antibodies allows monitoring of chaperonin complex dynamics without fixation artifacts. This approach requires:

  • Optimization of antibody concentration to prevent interference with native function

  • Careful control of injection parameters to minimize cell stress

  • Rapid imaging to capture transient interactions

• Complementary approaches: Combine FITC-conjugated CCT7 antibody labeling with:

  • Fluorescently tagged substrate proteins to monitor folding kinetics

  • FRAP (Fluorescence Recovery After Photobleaching) to measure chaperonin mobility

  • FLIM (Fluorescence Lifetime Imaging Microscopy) to detect conformational changes

• Quantitative analysis: Implement advanced image analysis techniques including:

  • Single-particle tracking of CCT7 complexes

  • Intensity fluctuation analysis to detect binding events

  • Colocalization coefficient calculation between CCT7 and substrate proteins

These approaches have revealed that CCT7 plays critical roles in stabilizing client proteins such as viral VP2, as demonstrated in cycloheximide chase experiments where CCT7 significantly improved VP2 protein stability .

What role does CCT7 play in viral replication, and how can FITC-conjugated antibodies elucidate this function?

CCT7's involvement in viral replication represents an emerging research area where FITC-conjugated antibodies provide valuable visualization capabilities. Research has demonstrated that:

• CCT7 directly interacts with viral proteins, particularly the VP2 protein of canine parvovirus (CPV) . The interaction region between CCT7 and VP2 is specifically located in the amino acids 231-320 region of VP2 .

• This interaction is functionally significant:

  • Cycloheximide chase experiments revealed that CCT7 improves VP2 protein stability

  • Knockdown of CCT7 expression by RNA interference significantly reduced VP2 protein expression

  • Overexpression of CCT7 increased viral copy numbers

• FITC-conjugated CCT7 antibodies enable visualization of these interactions through:

  • Colocalization studies showing spatial overlap between CCT7 and viral proteins

  • Time-course experiments tracking the dynamics of CCT7 recruitment during infection

  • Comparative analysis of CCT7 distribution in infected versus uninfected cells

The findings suggest that CCT7 functions as a proviral host factor by stabilizing viral proteins, potentially through its chaperonin activity facilitating proper folding of viral components .

How is CCT7 expression dysregulated in cancer, and what methodologies using FITC-conjugated antibodies can best characterize these changes?

CCT7 dysregulation in cancer represents a significant research area where FITC-conjugated antibodies provide valuable analytical capabilities. Research has established that:

These findings suggest CCT7 may promote cancer progression by enhancing tumor cell function, particularly in promoting myometrial invasion and metastasis .

What are the common causes of non-specific binding when using FITC-conjugated CCT7 antibodies, and how can these be mitigated?

Non-specific binding represents a significant challenge when using FITC-conjugated CCT7 antibodies. Common causes and solutions include:

Validation approaches should include:

• Testing on cell lines with confirmed CCT7 knockdown

• Peptide competition assays using the immunizing peptide

• Comparison with alternative CCT7 antibody clones

These strategies help ensure that the observed signals represent genuine CCT7 localization rather than technical artifacts.

How can researchers validate the specificity of FITC-conjugated CCT7 antibodies in their experimental systems?

Validating antibody specificity is critical for ensuring experimental rigor. For FITC-conjugated CCT7 antibodies, a comprehensive validation strategy includes:

• Genetic approaches:

  • siRNA knockdown: Transfect cells with CCT7-targeting siRNA (e.g., CCT7-Homo-914: 5′-CCACACAGUUGAGGAUUAUTT-3′, 5′-AUAAUCCUCAACUGUGUGGTT-3′) and verify signal reduction

  • CRISPR-Cas9 knockout: Generate CCT7-null cells as negative controls

  • Overexpression: Transfect cells with CCT7 expression vectors to verify signal enhancement

• Biochemical validation:

  • Western blot: Confirm single band of expected molecular weight

  • Immunoprecipitation followed by mass spectrometry

  • Peptide competition with the immunizing peptide sequence

• Cross-validation:

  • Compare staining patterns with alternative CCT7 antibody clones

  • Correlate with mRNA expression data

  • Multi-species reactivity testing based on epitope conservation

• Application-specific controls:

  • For immunofluorescence: Include secondary-only controls when testing unconjugated antibodies

  • For flow cytometry: Include fluorescence-minus-one (FMO) controls

  • For clinical samples: Include both positive and negative tissue controls

This systematic approach ensures that experimental observations genuinely reflect CCT7 biology rather than antibody artifacts .

What are the optimal parameters for detecting FITC-conjugated CCT7 antibodies using flow cytometry and confocal microscopy?

Optimizing detection parameters for FITC-conjugated CCT7 antibodies requires attention to the specific characteristics of FITC fluorescence:

Flow Cytometry Parameters:

Confocal Microscopy Settings:

For colocalization studies examining CCT7 interaction with client proteins (e.g., viral VP2), sequential scanning is recommended to prevent bleed-through between channels .

How can FITC-conjugated CCT7 antibodies be used to study the role of chaperonins in neurodegenerative diseases?

FITC-conjugated CCT7 antibodies offer valuable tools for investigating chaperonin dysfunction in neurodegenerative conditions. Research strategies include:

• Protein aggregation studies:

  • Visualize CCT7 association with aggregation-prone proteins (e.g., huntingtin, α-synuclein)

  • Quantify changes in CCT7 distribution in cellular models of neurodegeneration

  • Track CCT7 recruitment to inclusion bodies using time-lapse imaging

• Primary neuronal culture applications:

  • Compare CCT7 expression and localization between healthy and diseased neurons

  • Assess CCT7 colocalization with cytoskeletal elements critical for neuronal function

  • Evaluate changes in CCT7 distribution following cellular stress induction

• Patient-derived samples:

  • Analyze CCT7 expression in post-mortem brain tissue from patients with neurodegenerative diseases

  • Quantify CCT7 levels in patient-derived iPSC neurons carrying disease mutations

  • Correlate CCT7 expression patterns with disease severity markers

Research indicates that dysregulation of CCT7 has been linked to various diseases, including neurodegenerative disorders and cancer, highlighting its importance in cellular function . The chaperonin's role in proper protein folding makes it particularly relevant to conditions characterized by protein misfolding and aggregation.

What high-throughput screening approaches can utilize FITC-conjugated CCT7 antibodies to identify modulators of chaperonin function?

High-throughput screening (HTS) approaches using FITC-conjugated CCT7 antibodies can identify compounds that modulate chaperonin function:

• Automated microscopy platforms:

  • Design 384-well format assays measuring CCT7 redistribution upon compound treatment

  • Implement machine learning algorithms for pattern recognition in CCT7 localization

  • Quantify changes in CCT7-client protein colocalization following compound exposure

• Flow cytometry-based screens:

  • Develop multiplexed assays measuring CCT7 levels alongside client protein folding status

  • Implement bead-based technologies for simultaneous detection of multiple parameters

  • Utilize high-content flow cytometry to assess morphological changes alongside CCT7 status

• Functional readouts:

  • Design reporter systems where properly folded client proteins generate measurable signals

  • Monitor CCT7-dependent protein stability using pulse-chase approaches

  • Assess changes in CCT7 complex assembly using proximity-based assays

• Validation approaches:

  • Confirm hits using orthogonal assays (e.g., ELISA, Western blot)

  • Perform dose-response studies to establish potency

  • Assess specificity by examining effects on other chaperonin subunits

Research has shown that the HSF1A inhibitor can effectively downregulate CCT7 expression, demonstrating that chemical modulation of this pathway is feasible . This approach could identify novel compounds for both basic research and potential therapeutic development.

How can computational biology approaches complement experiments using FITC-conjugated CCT7 antibodies to understand chaperonin network dynamics?

Integrating computational biology with experimental data from FITC-conjugated CCT7 antibodies creates powerful approaches for understanding chaperonin networks: