pfl6 Antibody

What is the PFL6 antibody and what does it target?

PFL6 antibody (ab184984) is a rabbit polyclonal antibody developed against Prefoldin subunit 6 (PFDN6), a protein also known by alternative names including PFD6, HKE2, and Protein Ke2. This antibody specifically targets a recombinant fragment protein within Human PFDN6, spanning from amino acid 50 to the C-terminus. The target protein, Prefoldin subunit 6, functions as a component of the prefoldin complex, which plays an essential role in cytosolic protein folding mechanisms.

The prefoldin complex binds specifically to cytosolic chaperonin (c-CPN) and facilitates the transfer of target proteins. Additionally, PFDN6 interacts with nascent polypeptide chains to promote proper protein folding in environments where multiple competing pathways exist for nonnative proteins.

What are the key characteristics of the PFL6 antibody?

The key characteristics of the PFL6 antibody include:

This polyclonal nature provides a heterogeneous mixture of antibodies that recognize different epitopes on the target protein, potentially offering robust detection across various experimental conditions.

What validated applications exist for PFL6 antibody in research?

The PFL6 antibody has been validated for multiple experimental applications, making it a versatile tool for researchers investigating PFDN6 expression and localization. Based on extensive testing, the antibody has been confirmed to work effectively in the following applications with human samples:

• Immunohistochemistry with paraffin-embedded sections (IHC-P)

• Western blotting (WB)

• Immunocytochemistry/Immunofluorescence (ICC/IF)

Each application provides unique insights into the expression, localization, and interactions of PFDN6 in biological samples.

What are the optimal protocols for using PFL6 antibody?

Based on experimental validations, the following protocols have been established for optimal results with PFL6 antibody:

These protocols have been experimentally validated to produce reliable and reproducible results across different human tissue and cell samples.

How can I validate the specificity of PFL6 antibody in my experimental system?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For PFL6 antibody, consider implementing these methodological approaches:

• Western blot validation: The antibody should detect the predicted 14 kDa band in human cell lysates. Testing has confirmed specific detection in RT4 (human urinary bladder cancer cell line) and U-251 MG sp (human brain glioma cell line) whole cell lysates.

• Cross-reactivity testing: Compare staining patterns with other antibodies targeting the same protein or with genetic approaches (siRNA knockdown).

• Pattern validation: In immunofluorescence analyses, PFL6 antibody labels PFDN6 protein primarily at the nucleus and the Golgi apparatus in U-2 OS cells. This localization pattern should be consistent across appropriate cell types.

• Positive and negative controls: Include tissues or cell lines known to express or not express PFDN6 in your experiments.

How does protein fold-stability affect experimental outcomes when studying PFDN6?

Protein fold-stability represents an intrinsic feature that can significantly impact immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII) . When designing experiments involving PFDN6:

• Consider that the stability of PFDN6 may affect its processing by antigen-presenting cells and consequently the immune responses in immunological studies.

• Be aware that mutations in PFDN6 could alter its fold-stability, potentially affecting its function in protein folding pathways and experimental outcomes.

• The thermal stability of proteins can influence their resistance to endolysosomal proteases, which affects peptide presentation and subsequent immune responses .

Research on other proteins has demonstrated that in silico prediction of stabilizing or destabilizing point mutations can be useful for engineering proteins with altered stability for specific experimental purposes .

How can I detect and manage data contradictions when analyzing results from PFL6 antibody experiments?

When analyzing experimental data from antibody studies, contradictions may arise from various sources. A structured approach to identifying and resolving these contradictions includes:

• Define contradiction patterns: Consider contradiction patterns using the (α, β, θ) notation, where α represents the number of interdependent items, β is the number of contradictory dependencies, and θ is the minimal number of required Boolean rules to assess these contradictions .

• Implement systematic validation: Use multiple technical approaches (Western blot, IHC, ICC/IF) to verify findings and identify potential contradictions in PFDN6 detection.

• Cross-validation with other antibodies: When available, compare results with different antibodies targeting the same protein but recognizing different epitopes.

• Account for experimental variables: Consider how sample preparation methods, antibody concentration, incubation conditions, and detection systems may contribute to contradictory results .

This structured classification of contradiction checks allows for effective scoping of different contradiction patterns across multiple experiments and supports the implementation of a generalized contradiction assessment framework .

What insights can subcellular localization studies with PFL6 antibody provide about PFDN6 function?

Immunofluorescence analysis using the PFL6 antibody has revealed interesting subcellular localization patterns that provide valuable insights into the potential functions of PFDN6 beyond its established role in cytosolic protein folding:

• In U-2 OS (human bone osteosarcoma epithelial) cells, the antibody labeled PFL6 protein primarily at the nucleus and the Golgi apparatus when used at a concentration of 2 μg/ml.

• This localization pattern suggests potential roles for PFDN6 in:

  • Nuclear protein quality control

  • Protein trafficking through the Golgi apparatus

  • Potential involvement in secretory pathway protein folding

Further studies combining PFL6 antibody staining with markers for specific subcellular compartments could elucidate the precise roles of PFDN6 in these locations.

What are promising research areas for PFL6 antibody applications in disease models?

Given the fundamental role of molecular chaperones like PFDN6 in protein homeostasis, future research using PFL6 antibody might explore:

• Neurodegenerative disease research: Investigate expression patterns of PFDN6 in conditions characterized by protein misfolding (e.g., Alzheimer's, Parkinson's).

• Cancer biology: Examine potential alterations in PFDN6 expression and localization in various cancer types, especially those characterized by proteostasis imbalance.

• Stress response pathways: Study how cellular stress affects PFDN6 expression, localization, and function using the antibody to track changes under different stress conditions.

• Developmental biology: Investigate the expression pattern of PFDN6 during development, especially in tissues with high protein synthesis rates.

How can advanced antibody development approaches improve PFL6 antibody specificity?

Recent research on antibody development highlights advanced techniques that could potentially enhance PFL6 antibody specificity:

• Alternative host species: Using chickens instead of mammals to bypass immune tolerance for highly conserved proteins.

• Comprehensive deselection strategies: Implementing deselection against closely related proteins to improve specificity.

• Epitope mapping: Utilizing atomic-level epitope mapping to understand the structural basis of antibody specificity.

• Recombinant antibody technology: Developing single-chain variable fragments (scFvs) or antigen-binding fragments (Fabs) with enhanced specificity for PFDN6.

What are common issues when using PFL6 antibody in different applications?

When working with PFL6 antibody, researchers may encounter several common challenges:

• Non-specific binding: May occur particularly in Western blotting applications. Optimize blocking conditions and antibody dilutions to reduce background.

• Variable signal intensity: Different tissue types may exhibit variable staining intensity. Validate the antibody in your specific sample type before proceeding with full experiments.

• Epitope masking: Fixation methods may affect epitope accessibility. Consider testing different antigen retrieval methods for IHC applications.

• Batch-to-batch variability: Polyclonal antibodies like PFL6 can exhibit some variability between production lots. Include appropriate positive controls with each experiment.

Systematic troubleshooting approaches that carefully control variables such as antibody concentration, incubation conditions, and sample preparation methods can help resolve these issues.

How can I assess data quality and identify potential contradictions in PFL6 antibody experiments?

Establishing rigorous quality control measures is essential for generating reliable data with PFL6 antibody:

• Control samples: Always include positive and negative controls to validate antibody performance in each experiment.

• Technical replicates: Perform multiple technical replicates to identify potential variability in antibody performance.

• Orthogonal validation: Validate findings using multiple detection methods (e.g., WB, IHC, and IF) to ensure consistency across platforms.

• Contradiction analysis: Apply structured contradiction analysis using Boolean logic to identify impossible combinations of values in interdependent data items . This approach is particularly valuable when integrating multiple datasets or experimental approaches.

By implementing these quality control measures, researchers can improve the reliability and reproducibility of their experiments using PFL6 antibody.