PFDN1 Antibody Pair

What is PFDN1 and why is it an important research target?

PFDN1 (Prefoldin subunit 1) is a co-chaperone protein primarily known for its classic cytoplasmic functions in the folding of actin and tubulin monomers during cytoskeletal assembly . Recent research has revealed additional roles for PFDN1, including its involvement in the epithelial-mesenchymal transition (EMT), cancer progression, and co-transcriptional pre-mRNA splicing .

PFDN1 is part of the prefoldin complex, which consists of six subunits forming a double beta barrel assembly with protruding coiled-coils that bind and stabilize newly synthesized polypeptides . Studying PFDN1 is particularly important because:

• It has been implicated in multiple cancer types, including lung cancer and gastric cancer

• It plays roles in both cytoplasmic protein folding and nuclear gene regulation

• Its dysregulation has been linked to protein misfolding diseases and cancer progression

How do PFDN1 antibody pairs differ from single antibodies in research applications?

PFDN1 antibody pairs consist of two antibodies designed to work together in sandwich-based detection techniques:

• The capture antibody binds to PFDN1 and immobilizes it on a solid surface

• The detection antibody (often biotinylated) binds to a different epitope of the captured PFDN1 protein

This paired approach offers several advantages compared to single antibody detection:

• Higher specificity due to the requirement for two separate binding events

• Improved sensitivity through signal amplification

• Better quantification capabilities, especially in complex biological samples

• Reduced background signals and false positives

For instance, the PFDN1 antibody pair available from Cusabio (CSB-EAP01485) includes specific capture and detection antibodies designed for sandwich ELISA applications, with reactivity to PFDN1 across multiple species including human, rat, and mouse .

What are the optimal conditions for using PFDN1 antibody pairs in sandwich ELISA?

Based on research protocols and commercial recommendations, optimal conditions for PFDN1 antibody pairs in sandwich ELISA include:

As noted in the Cusabio product documentation: "We recommend using the capture antibody at a concentration of 0.5 μg/ml and the detection antibody at a concentration of 0.5 μg/ml. Optimal dilutions should be determined experimentally by the researcher."

The effectiveness of the ELISA can be validated using positive control samples such as cell lysates from HT-29, PC-12, or mouse brain tissue, which are known to express PFDN1 .

How should I design experiments to study PFDN1's role in cancer progression?

Based on published methodologies, a comprehensive experimental approach to studying PFDN1's role in cancer progression should include:

• Expression analysis in clinical samples:

  • Compare PFDN1 levels in tumor vs. adjacent normal tissues using qRT-PCR, Western blot, and immunohistochemistry

  • Correlate PFDN1 expression with clinical parameters including metastasis status and patient survival

• Functional studies using genetic manipulation:

  • Overexpression: Transfect cells with PFDN1-expressing plasmids (e.g., CMV-driven expression vectors with appropriate tags)

  • Knockdown: Use siRNA targeting PFDN1 (validated sequences are available in the literature)

  • Knockout: Generate PFDN1-null cell lines using CRISPR-Cas9 (gRNA design can be modeled after published sequences)

• Phenotypic assays:

  • Cell migration: Wound-healing assay

  • Invasion: Transwell assay

  • EMT markers: Western blot analysis of E-cadherin, N-cadherin, and other markers

  • Cell cycle analysis: Flow cytometry

  • In vivo metastasis: Xenograft models in nude mice

As demonstrated in a study on gastric cancer: "PFDN1 levels were significantly upregulated in GC tissues compared with those in matched adjacent normal tissues. PFDN1 upregulation correlated strongly with clinical metastasis and unfavorable prognosis for GC patients. In vitro and in vivo studies revealed that PFDN1 facilitated GC cell migration, invasion and metastasis."

How can I investigate the nuclear functions of PFDN1 in transcription and splicing?

Recent research has revealed that PFDN1 plays important roles in the nucleus, including effects on transcription and pre-mRNA splicing. To investigate these functions:

• Subcellular localization studies:

  • Perform nuclear/cytoplasmic fractionation followed by Western blotting

  • Use immunofluorescence microscopy with PFDN1 antibodies alongside nuclear markers

  • Consider using tagged versions (GFP-PFDN1) for live-cell imaging

• Transcription analysis:

  • Chromatin immunoprecipitation (ChIP) assays to identify PFDN1 binding to specific promoters

  • RNA-seq or qRT-PCR analysis after PFDN1 knockdown/overexpression

  • Use of transcription inhibitors (e.g., DRB) to study kinetics

• Splicing analysis:

  • RT-PCR with primers designed to detect specific splicing events

  • Analysis of precursor RNA containing specific introns

  • Co-immunoprecipitation with splicing factors (e.g., U2AF65, PRPF19)

A published study utilized this approach: "To study both transcription and co-transcriptional splicing, primer pairs were designed to amplify the cDNA derived from different kinds of unspliced... Using the same samples, we measured co-transcriptional splicing by the appearance of precursors containing intron 6 of CTNNBL1, but lacking intron 5, already spliced."

What are the best methods to study the interaction between PFDN1 and the TGF-β1 signaling pathway?

Research has identified connections between PFDN1 and TGF-β1 signaling, particularly in the context of EMT and cancer progression. To investigate this interaction:

• Induction studies:

  • Treat cells with TGF-β1 at varying concentrations (1-10 ng/ml) and time points

  • Monitor PFDN1 expression changes via Western blot and qRT-PCR

  • Analyze EMT markers simultaneously

• Signaling pathway analysis:

  • Use inhibitors of TGF-β1 receptors or downstream mediators

  • Knockdown key TGF-β1 signaling components (Smad2, Smad3) to determine effects on PFDN1 expression

  • Assess phosphorylation status of signaling molecules

• Functional rescue experiments:

  • Perform PFDN1 knockdown followed by TGF-β1 treatment

  • Overexpress PFDN1 in the presence of TGF-β1 pathway inhibitors

  • Analyze downstream targets like cyclin A

This approach is supported by research findings: "Further experiments showed that knockdown of either smad2 or smad3 inhibits TGF-β1-induced EMT and expression level changes of PFDN1 and cyclin A, confirming the involvement of TGF-β1 signaling effectors."

What are common issues when using PFDN1 antibody pairs and how can they be resolved?

PFDN1 antibodies have been validated to detect a protein with an observed molecular weight of 14 kDa, consistent with its calculated molecular weight . If unexpected bands are observed, consider additional validation steps.

How do I interpret contradictory results about PFDN1's role in different cancer types?

Research has revealed that PFDN1 may have context-dependent roles in different cancer types or experimental conditions. When facing contradictory results:

• Consider tissue/cell type specificity:

  • PFDN1 expression and function may vary across different tissues and cancer types

  • Verify expression patterns in your specific experimental system

• Analyze experimental context:

  • In vitro vs. in vivo models might show different outcomes

  • Acute (siRNA) vs. chronic (stable knockdown) loss of function may have divergent effects

  • Consider the potential compensatory mechanisms that may emerge in different models

• Examine pathway interactions:

  • PFDN1's effects may depend on the status of related pathways (TGF-β, Wnt/β-catenin)

  • Perform pathway inhibition studies in parallel with PFDN1 manipulation

• Validation in multiple systems:

  • Use different cell lines representing the same cancer type

  • Validate findings in patient-derived samples

  • Employ multiple methodologies (e.g., different knockdown approaches)

Research has shown that "PFDN1 modulated GC cell behavior by activating Wnt/β-catenin signaling-mediated EMT" , while in lung cancer, "these effects were mediated through the transcriptional inhibition of cyclin A by PFDN1" , indicating potentially different molecular mechanisms in different cancer contexts.

What are the emerging applications of PFDN1 antibody pairs beyond traditional research techniques?

Recent advances suggest several innovative applications for PFDN1 antibody pairs:

• Multiplex protein detection systems:

  • Integration into multiplex platforms to simultaneously detect PFDN1 alongside other cancer biomarkers

  • Development of microarray-based detection systems for high-throughput screening

• In vivo imaging applications:

  • Conjugation with imaging agents for non-invasive detection of PFDN1-expressing tumors

  • Development of multimodal imaging probes combining detection with therapeutic targeting

• Single-cell analysis techniques:

  • Adaptation for flow cytometry to study heterogeneity of PFDN1 expression in tumors

  • Integration with mass cytometry for simultaneous detection of multiple parameters

• Liquid biopsy applications:

  • Development of sensitive detection methods for PFDN1 in circulating tumor cells or exosomes

  • Correlation with tumor burden and treatment response

These applications build upon the established specificity of PFDN1 antibody pairs and their validated use in detecting this important cancer-associated protein.

How can PFDN1's dual roles in protein folding and transcriptional regulation be experimentally separated?

To dissect PFDN1's cytoplasmic and nuclear functions:

• Domain-specific mutants:

  • Generate mutants that disrupt specific functions (e.g., protein folding vs. DNA binding)

  • Create localization-specific variants with nuclear localization or export signals

  • Perform rescue experiments with these mutants in PFDN1-knockdown cells

• Interactome analysis:

  • Perform immunoprecipitation coupled with mass spectrometry in different cellular compartments

  • Identify compartment-specific interaction partners

  • Validate key interactions that may mediate specific functions

• Temporal control of PFDN1 activity:

  • Use inducible expression systems to control timing of PFDN1 expression/depletion

  • Employ optogenetic approaches to regulate PFDN1 activity in specific cellular compartments

  • Analyze immediate vs. delayed effects to distinguish direct from indirect consequences

• High-resolution microscopy:

  • Track PFDN1 dynamics using super-resolution microscopy

  • Correlate localization with functional outcomes in real-time

  • Employ FRAP (Fluorescence Recovery After Photobleaching) to study mobility in different compartments