PFDN4 Antibody

What are the most common applications for PFDN4 antibodies in research?

PFDN4 antibodies are primarily utilized in the following research applications:

• Western blotting (WB): Used for detecting and quantifying PFDN4 protein in cell or tissue lysates with recommended dilutions of 1:200-1:2000 .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Used for cellular localization studies with recommended dilutions of 1:50-1:200 .

• ELISA (Enzyme-Linked Immunosorbent Assay): Used for quantitative detection of PFDN4 in various sample types including serum, plasma, cell culture supernatant, and tissue homogenates .

These applications enable researchers to investigate PFDN4 expression levels, localization patterns, and interactions with other proteins in both normal and pathological conditions.

How do I select the appropriate PFDN4 antibody for my specific experiment?

When selecting a PFDN4 antibody for your research, consider the following factors:

Always review the validation data for your specific application and species before proceeding with experiments. For instance, PFDN4 antibody CAB15300 has been validated with multiple positive samples including DU145, HeLa, 293T, MCF7, and mouse kidney tissue .

What is the optimal protocol for Western blot detection of PFDN4?

For optimal Western blot detection of PFDN4:

• Sample preparation:

  • Lyse cells or tissues in appropriate buffer containing protease inhibitors

  • Denature proteins at 95°C for 5 minutes in sample buffer

  • Load 20-40 μg of total protein per lane

• Gel electrophoresis:

  • Use 12-15% SDS-PAGE (given PFDN4's calculated MW of 15 kDa)

  • Include molecular weight markers

• Transfer and blocking:

  • Transfer to PVDF or nitrocellulose membrane

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute PFDN4 antibody to 1:200-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

• Secondary antibody and detection:

  • Wash membrane 3-4 times with TBST

  • Incubate with HRP-conjugated secondary antibody against rabbit IgG

  • Develop using ECL substrate and appropriate imaging system

Expected result: A specific band at approximately 15 kDa corresponding to PFDN4 .

How should I optimize immunofluorescence protocols for PFDN4 detection?

For optimal immunofluorescence detection of PFDN4:

• Sample preparation:

  • Culture cells on coverslips or prepare tissue sections (5-8 μm thick)

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 for 10 minutes

• Blocking:

  • Block with 5% normal serum (from the species of secondary antibody) in PBS for 1 hour

• Antibody incubation:

  • Dilute PFDN4 primary antibody at 1:50-1:200 in blocking solution

  • Incubate overnight at 4°C in a humidified chamber

  • Wash 3x with PBS

• Secondary antibody and counterstaining:

  • Apply fluorophore-conjugated anti-rabbit secondary antibody (1:200-1:1000)

  • Counterstain nuclei with DAPI

  • Mount with anti-fade mounting medium

• Visualization:

  • Observe subcellular localization using confocal or fluorescence microscopy

  • Expect to see primarily cytoplasmic staining with some mitochondrial and nuclear localization

What sample preparation methods are recommended for PFDN4 ELISA assays?

Different sample types require specific preparation methods for optimal PFDN4 detection by ELISA:

• Serum preparation:

  • Collect blood in serum separator tubes

  • Allow samples to clot for 2 hours at room temperature or overnight at 2-8°C

  • Centrifuge at approximately 1000 × g (or 3000 rpm) for 15 minutes

  • Carefully remove serum and assay immediately or store aliquots at -20°C or -80°C

• Plasma preparation:

  • Collect blood using EDTA or heparin as anticoagulant

  • Centrifuge samples for 15 minutes at 1000 × g (3000 rpm) at 2-8°C within 30 minutes of collection

  • Carefully remove plasma and assay immediately or store aliquots at -20°C or -80°C

• Tissue homogenate preparation:

  • Rinse tissues thoroughly in ice-cold PBS (0.02 mol/L, pH 7.0-7.2)

  • Weigh tissue before homogenization

  • Mince tissues into small pieces

  • Homogenize in an appropriate volume of PBS using a glass homogenizer on ice

  • Subject the suspension to ultrasonication or two freeze-thaw cycles

  • Centrifuge for 15 minutes at 1500 × g (5000 rpm)

  • Collect supernatant for immediate analysis or store at -20°C or -80°C

• Cell culture supernatant:

  • Collect medium after appropriate treatment period

  • Centrifuge at 3000 rpm for 10 minutes to remove cells and debris

  • Use immediately or aliquot and store at -20°C or -80°C

How do I evaluate PFDN4 antibody specificity in my experiments?

To evaluate and confirm PFDN4 antibody specificity:

• Positive controls:

  • Include known PFDN4-expressing samples such as DU145, HeLa, 293T, or MCF7 cell lysates, or mouse kidney tissue

  • Compare your experimental samples against these validated positive controls

• Molecular weight verification:

  • Confirm the detected band appears at the expected molecular weight (~15 kDa)

  • Be aware that post-translational modifications may cause slight shifts in apparent molecular weight

• Blocking peptide competition:

  • Perform a parallel experiment with antibody pre-incubated with the immunizing peptide

  • A specific signal should be significantly reduced or eliminated in the blocked sample

• Knockdown/knockout validation:

  • Compare signal between wild-type samples and those with PFDN4 knockdown or knockout

  • Specific signals should be reduced or absent in knockdown/knockout samples

• Cross-reactivity assessment:

  • If working across species, verify specificity in each organism

  • Compare observed staining patterns with published literature on PFDN4 localization (cytoplasm, mitochondria, nucleus)

What quantification methods are most appropriate for PFDN4 expression analysis?

Depending on your experimental approach, consider these quantification methods:

• Western blot densitometry:

  • Normalize PFDN4 signal to loading controls (β-actin, GAPDH, tubulin)

  • Use image analysis software (ImageJ, Image Lab, etc.) to quantify band intensity

  • Report results as relative expression compared to control samples

• Immunofluorescence quantification:

  • Measure mean fluorescence intensity within defined cellular regions

  • Quantify the percentage of cells showing positive staining

  • Analyze co-localization with other markers using Pearson's correlation coefficient

• ELISA quantification:

  • Generate standard curves using purified PFDN4 protein standards

  • Ensure samples fall within the detection range (e.g., 250-5000 pg/mL for some kits)

  • Calculate concentration using regression analysis of standard curve

  • Multiply by any dilution factors used during sample preparation

• Statistical analysis:

  • Perform appropriate statistical tests (t-test, ANOVA) to determine significance

  • Report results with p-values and confidence intervals

  • Include biological replicates (n≥3) to ensure reproducibility

How can I investigate PFDN4 interactions with other prefoldin subunits and client proteins?

To study PFDN4 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use PFDN4 antibody to pull down PFDN4 and associated proteins

  • Analyze precipitated complexes by Western blot or mass spectrometry

  • Look for other prefoldin subunits and known client proteins

  • Use epitope-tagged PFDN4 constructs as an alternative approach

• Proximity ligation assay (PLA):

  • Use PFDN4 antibody together with antibodies against potential interacting partners

  • Visualize protein-protein interactions in situ with subcellular resolution

  • Quantify interaction signals using appropriate image analysis software

• Fluorescence resonance energy transfer (FRET):

  • Generate fluorescent protein fusions with PFDN4 and potential partners

  • Measure energy transfer between fluorophores as evidence of protein proximity

  • Analyze FRET efficiency in different cellular compartments and conditions

• Bimolecular fluorescence complementation (BiFC):

  • Split a fluorescent protein between PFDN4 and potential interacting proteins

  • Reconstitution of fluorescence indicates physical interaction between proteins

  • Allows visualization of interaction sites within cells

• Proteomics approaches:

  • Perform PFDN4 immunoprecipitation followed by mass spectrometry

  • Compare interactome under different cellular conditions (stress, differentiation)

  • Validate key interactions using orthogonal methods

What experimental approaches are suitable for studying PFDN4's role in protein misfolding diseases?

To investigate PFDN4's involvement in protein misfolding diseases:

• Disease model systems:

  • Generate PFDN4 knockdown/knockout in cellular or animal models of neurodegenerative diseases

  • Assess effects on protein aggregation, cellular toxicity, and disease progression

  • Rescue experiments by re-introducing wild-type PFDN4

• Protein aggregation assays:

  • Monitor aggregation of disease-associated proteins (e.g., huntingtin, α-synuclein, tau) in the presence or absence of functional PFDN4

  • Use fluorescence-based aggregation assays, filter trap assays, or sedimentation approaches

  • Correlate PFDN4 levels with aggregation kinetics

• Patient sample analysis:

  • Compare PFDN4 expression, localization, and modifications in patient vs. control samples

  • Use immunohistochemistry to examine co-localization with disease-specific aggregates

  • Perform Western blot analysis to quantify PFDN4 levels in affected tissues

• Stress response studies:

  • Examine how cellular stressors affect PFDN4 expression and function

  • Investigate whether PFDN4 overexpression can protect against proteotoxic stress

  • Monitor chaperone network compensatory mechanisms in PFDN4-deficient cells

How can I assess the impact of post-translational modifications on PFDN4 function?

To study post-translational modifications (PTMs) of PFDN4:

• Identification of PTMs:

  • Immunoprecipitate PFDN4 followed by mass spectrometry analysis

  • Use phospho-specific, acetylation-specific, or ubiquitin-specific antibodies

  • Compare PTM patterns under different cellular conditions

• Site-directed mutagenesis:

  • Generate PFDN4 constructs with mutations at potential PTM sites

  • Express mutants in cells and assess effects on:

    • Protein stability and half-life

    • Subcellular localization

    • Interaction with other prefoldin subunits and clients

    • Chaperone activity

• PTM enzyme modulation:

  • Inhibit or activate enzymes responsible for specific PTMs

  • Assess effects on PFDN4 function and protein folding capacity

  • Use siRNA to knockdown specific kinases, phosphatases, or other modifying enzymes

• Functional assays:

  • Compare wild-type and PTM-mutant PFDN4 in protein folding assays

  • Assess ability to prevent aggregation of model substrates

  • Measure interaction with cytoskeletal components and influence on cellular architecture

Why might I observe weak or no signal when using PFDN4 antibodies in Western blot?

How can I reduce background in PFDN4 immunofluorescence experiments?

To reduce background in PFDN4 immunofluorescence:

• Optimize fixation and permeabilization:

  • Test different fixatives (PFA, methanol, acetone)

  • Adjust permeabilization conditions (concentration and time)

  • Ensure complete washing between steps

• Improve blocking:

  • Increase blocking time (2-3 hours at room temperature)

  • Try different blocking agents (BSA, normal serum, commercial blockers)

  • Add 0.1-0.3% Triton X-100 to blocking solution

• Antibody optimization:

  • Titrate primary antibody (start with 1:50-1:200 dilution)

  • Pre-absorb antibody with cell/tissue lysate from non-relevant species

  • Increase washing steps after antibody incubations

• Controls:

  • Include secondary-only controls to assess non-specific binding

  • Use isotype controls at matching concentrations

  • Include PFDN4 knockdown samples as negative controls

• Image acquisition:

  • Optimize exposure settings using control samples

  • Use appropriate filter sets to minimize autofluorescence

  • Apply image processing consistently across all samples

What factors affect PFDN4 ELISA assay reproducibility and how can they be controlled?

To improve ELISA reproducibility when detecting PFDN4:

• Sample handling:

  • Maintain consistent sample collection and processing procedures

  • Avoid repeated freeze-thaw cycles of samples

  • Ensure samples are within the detection range (250-5000 pg/mL)

• Assay conditions:

  • Maintain consistent incubation times and temperatures

  • Use a plate shaker during incubations for uniform binding

  • Ensure complete washing between steps without allowing wells to dry

• Reagent quality:

  • Use freshly prepared reagents whenever possible

  • Store components according to manufacturer recommendations

  • Check expiration dates on all kit components

  • Prepare standard curves freshly for each assay

• Technical considerations:

  • Use calibrated pipettes and verify their accuracy regularly

  • Run all samples and standards in duplicate or triplicate

  • Include internal control samples across multiple plates/experiments

  • Equilibrate all reagents to room temperature before use

• Data analysis:

  • Use appropriate curve-fitting methods for standard curves

  • Apply consistent analysis parameters across experiments

  • Report both intra-assay and inter-assay coefficients of variation