PDLIM5 Antibody, Biotin conjugated

What is PDLIM5 and what cellular roles does it play?

PDLIM5 (PDZ and LIM domain protein 5), also known as Enigma homolog (ENH), is a 63 kDa cytoplasmic protein composed of a PDZ domain in the N-terminus and three consecutive LIM domains in the C-terminus. It was first discovered in 1996 by Kuroda et al. using yeast two-hybrid technique with PKC as bait protein . PDLIM5 is anchored to the actin cytoskeleton through its PDZ domain and recruits actin filament-associated proteins, which contributes to cytoskeletal organization, cell lineage specification, organ development, and oncogenesis .

PDLIM5 has multiple tissue-specific functions:

• In cardiac tissue: Plays an important role in heart development by scaffolding PKC to the Z-disk region and regulating cardiomyocyte expansion. Overexpression promotes heart hypertrophy development .

• In neurons: Contributes to dendritic spine morphogenesis and may be required to restrain postsynaptic growth of excitatory synapses. Isoform 1 expression favors spine thinning and elongation .

• In cancer: Facilitates cell tumorigenesis, particularly in prostate cancer through regulation of migration and invasion .

• In vascular smooth muscle cells: Regulates TGF-β signaling pathways and inhibits COL1A2 expression .

What applications are suitable for PDLIM5 Antibody, Biotin conjugated?

PDLIM5 Antibody, Biotin conjugated has been validated for several applications, with varying levels of optimization:

Note: While biotin conjugation makes this antibody particularly useful for detection systems utilizing avidin/streptavidin, researchers should consider potential background issues from endogenous biotin in tissues .

How should PDLIM5 Antibody, Biotin conjugated be stored for optimal stability?

For maximum stability and activity retention, PDLIM5 Antibody, Biotin conjugated should be stored according to these guidelines:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles by preparing aliquots for single use

• For some formulations containing 50% glycerol, aliquoting may be unnecessary for -20°C storage

• If shipping is required, use blue ice (4°C) conditions for short durations

• Buffer composition (typically PBS with preservatives like 0.03% Proclin 300 and 50% glycerol, pH 7.4) helps maintain stability

Long-term stability is typically guaranteed for one year after shipment when stored properly .

How does PDLIM5 contribute to cancer progression and what methodologies are best for studying this relationship?

PDLIM5 has been identified as an oncogenic factor, particularly in prostate cancer (PCa). Research methodologies to study this relationship include:

Expression Analysis:

• PDLIM5 is abnormally upregulated in PCa tissues compared to normal prostate tissue

• Oncomine microarray data mining shows PDLIM5 overexpression correlates with:

  • Higher Gleason score (P=0.009)

  • Capsular invasion (P=0.009)

  • Extracapsular extension (P=0.056)

  • Biochemical recurrence (BCR) (P=0.054)

Functional Analysis Approaches:

Recommended methodological approaches:

• qPCR and Western blotting for expression analysis in cell lines and tissues

• Plate colony formation assay and MTT for proliferation assessment

• FACS for cell cycle distribution and apoptosis evaluation

• Migration and invasion assays for metastatic potential assessment

• Xenograft nude mouse models for in vivo confirmation

What is the role of PDLIM5 in TGFβ signaling and what techniques can detect these interactions?

PDLIM5 functions as a regulator of TGFβ signaling through multiple mechanisms:

PDLIM5-Smad Interactions:

• PDLIM5 interacts with Smad2/3, key mediators of TGFβ signaling

• Under basal conditions, PDLIM5 forms a protein complex with Smad2, which is disrupted by TGFβ treatment

• PDLIM5 stabilizes SMAD3 by counteracting STUB1-mediated degradation

Experimental Approaches to Study These Interactions:

• RNA Immunoprecipitation and RNA Pull-Down Assays:

  • Can demonstrate specific interactions between circRNAs like circZBTB46 and PDLIM5

• Co-Immunoprecipitation (Co-IP):

  • Reciprocal Co-IP assays can reveal time-dependent changes in PDLIM5-Smad2 interactions

  • Treatment with TGFβ (5 ng/mL) leads to a time-dependent decrease in Smad2 levels in anti-PDLIM5 immunoprecipitates

• Overexpression and Knockdown Studies:

  • Adenoviral expression vectors (Ad-PDLIM5) can be used for overexpression

  • siRNA can be used for knockdown

  • Effects on Smad2 phosphorylation and downstream gene expression (e.g., COL1A2) can be measured by Western blot

• FRET (Fluorescence Resonance Energy Transfer):

  • Acceptor bleach FRET experiments with CFP-SMAD3 as donor and YFP-STUB1 as acceptor can verify protein interactions

  • PDLIM5 overexpression disrupts FRET between SMAD3 and STUB1, confirming that PDLIM5 represses STUB1-SMAD3 interaction

• IP-MS (Immunoprecipitation-Mass Spectrometry):

  • Can identify binding partners of PDLIM5, including E3 ligases like STUB1

What technical considerations should be addressed when using biotin-conjugated antibodies in tissues with endogenous biotin?

Endogenous biotin in tissues presents significant challenges when using biotin-conjugated antibodies due to potential false-positive signals. Several technical considerations and solutions are available:

Source of Background:

• Endogenously biotinylated proteins with carboxylase activity are found in mitochondria

• These can produce substantial background signals when avidin-biotin detection systems are used

Technical Solutions:

• Pre-blocking Endogenous Biotin:

  • Use streptavidin and biotin solutions to block endogenous biotin before applying biotinylated antibodies

  • Commercial kits such as the Endogenous Biotin-Blocking Kit are available

• Buffer Optimization:

  • Non-specific binding of avidin conjugates to nitrocellulose can be blocked more effectively by adding extra salts to buffers rather than using protein-based blocking reagents

• Alternative Detection Strategies:

  • Consider using DSB-X biotin technology, which allows for reversible binding between biotin and avidin/streptavidin

  • This technology enables stripping of the avidin-biotin complex without affecting other immunolabeling components

• Validation Controls:

  • Include appropriate negative controls without primary antibody but with detection reagents

  • Include tissue sections known to have high endogenous biotin (e.g., liver, kidney) as positive controls for blocking effectiveness

• Alternative Conjugation:

  • Consider using directly conjugated antibodies (e.g., fluorophore-labeled) instead of biotin-conjugated antibodies when working with tissues known to have high endogenous biotin

How can researchers optimize PDLIM5 knockdown experiments for studying its function in different cellular contexts?

Optimizing PDLIM5 knockdown experiments requires careful consideration of several technical aspects:

Vector Selection and Delivery:

• Lentivirus-mediated short hairpin RNA (shRNA) systems have been successfully used for PDLIM5 knockdown

• Ensure transduction efficiency of ~90% (can be verified by GFP expression if using a GFP reporter)

• For cell-specific effects, consider using cell lines that express high levels of PDLIM5 (e.g., DU145 and PC-3 for prostate cancer studies)

Knockdown Validation:

• Confirm knockdown efficiency using multiple techniques:

  • Western blot (protein level)

  • qRT-PCR (mRNA level)

  • Target 60-80% reduction for optimal phenotype observation without cellular toxicity

Control Selection:

• Use non-targeting shRNA/siRNA controls with similar GC content

• Include wild-type and mock-transfected controls

• For rescue experiments, use expression vectors containing PDLIM5 coding sequences with silent mutations that resist knockdown

Functional Assays Based on Cell Type:

• For Cancer Cells:

  • Colony formation assays

  • MTT for proliferation

  • FACS for cell cycle analysis and apoptosis

  • Migration and invasion assays

• For Vascular Smooth Muscle Cells:

  • TGFβ signaling assays (measuring Smad2 phosphorylation)

  • COL1A2 expression analysis

  • Cell adhesion to fibronectin

  • Vasculogenic mimicry assays

• For Cardiac/Neuronal Cells:

  • Cytoskeletal organization analysis

  • Synapse morphology analysis

  • Electrophysiological measurements

Timing Considerations:

• Allow 48-72 hours post-transfection for optimal knockdown

• For signaling studies (e.g., with TGFβ), determine optimal stimulation time through time-course experiments

• For cell cycle effects, synchronize cells before knockdown

What are the critical factors for designing experiments to detect PDLIM5 isoforms using biotin-conjugated antibodies?

PDLIM5 exists in multiple isoforms (including ENH1-4), making experimental design challenging. Critical factors include:

Antibody Selection Considerations:

• Determine if the antibody recognizes all or specific isoforms

• The antibody epitope location is crucial - some PDLIM5 antibodies target regions present in all isoforms, while others may be isoform-specific

• The full-length isoform (ENH1) is widely expressed, while shorter isoforms lacking LIM motifs have tissue-specific expression patterns (cardiac: ENH3; skeletal muscle: ENH2, ENH3, ENH4)

Sample Preparation:

• For protein extraction, consider tissue-specific protocols as PDLIM5 associates with cytoskeleton

• Use multiple lysis buffers to ensure complete extraction (standard RIPA buffer may not fully extract cytoskeleton-associated proteins)

• Include protease inhibitors to prevent degradation

Detection System Optimization:

• For Western blot: Use gradient gels (4-12%) to resolve multiple isoforms

• Expected molecular weights:

  • Full-length PDLIM5: 63-69 kDa

  • Shorter isoforms: May range from 30-50 kDa

• For biotin-conjugated antibodies, use streptavidin-HRP with optimized concentration

• Consider sample-dependent dilution optimization

Controls and Validation:

• Include positive controls (tissues/cells known to express specific isoforms):

  • Heart tissue for ENH1/ENH3

  • Skeletal muscle for ENH2/3/4

  • Brain tissue for full-length isoform

• Consider using cell lines with PDLIM5 knockout as negative controls

• For isoform-specific detection, validate with recombinant proteins of each isoform

How should researchers interpret and troubleshoot contradictory data regarding PDLIM5's role in TGFβ signaling pathways?

Studies have revealed seemingly contradictory roles for PDLIM5 in TGFβ signaling, requiring careful experimental design and data interpretation:

Observed Contradictions:

• Role in SMAD3 Regulation:

  • Some studies show PDLIM5 stabilizes SMAD3 by preventing STUB1-mediated degradation

  • Other research indicates PDLIM5 inhibits TGFβ-induced Smad signaling and COL1A2 expression

• Cell Type Differences:

  • In vascular smooth muscle cells (VSMCs), PDLIM5 inhibits TGFβ-Smad signaling

  • In non-small cell lung cancer (NSCLC), PDLIM5 enhances TGFβ signaling

Methodological Approaches to Resolve Contradictions:

• Context-Dependent Analysis:

  • Perform experiments in multiple cell types simultaneously

  • Compare primary cells vs. cell lines

  • Analyze normal vs. disease states (e.g., cancer vs. normal tissues)

• Mechanistic Validation:

  • Examine upstream regulators (e.g., circZBTB46) across different cell types

  • Use domain mapping to identify which PDLIM5 domains are responsible for different effects

  • Determine if post-translational modifications of PDLIM5 differ between contexts

• Time-Course Analysis:

  • Perform detailed time-course experiments after TGFβ stimulation

  • Short-term vs. long-term effects may differ

  • Monitor dynamic changes in protein complexes (PDLIM5-SMAD3-STUB1)

• Quantitative Assessment:

  • Use quantitative proteomics to measure SMAD3 half-life in different contexts

  • Analyze phosphorylation status of SMADs at multiple time points

  • Measure ratios of PDLIM5:SMAD3:STUB1 to determine threshold effects

Troubleshooting Approaches:

• Use multiple knockdown/overexpression techniques to ensure consistency

• Validate antibody specificity with recombinant proteins

• Consider genetic background differences in cell lines

• Account for feedback mechanisms in the TGFβ pathway

• Examine effects on multiple downstream targets, not just a single readout

What are the best practices for quantifying and analyzing PDLIM5 expression in tissue samples using biotin-conjugated antibodies?

Accurate quantification of PDLIM5 expression in tissues requires attention to several methodological considerations:

Sample Preparation:

• Fresh tissues should be promptly fixed (10% neutral buffered formalin) or flash-frozen

• For FFPE tissues, optimize antigen retrieval methods:

  • TE buffer pH 9.0 is recommended for PDLIM5 detection

  • Alternatively, citrate buffer pH 6.0 may be used

• Section thickness should be consistent (typically 4-5 μm for IHC)

Staining Protocol Optimization:

• Blocking Endogenous Biotin:

  • Critical step for tissues with high endogenous biotin (e.g., liver, kidney, brain)

  • Use avidin-biotin blocking kits before applying primary antibody

• Antibody Dilution:

  • Determine optimal dilution through titration (typically 1:50-1:500 for IHC)

  • Include both positive control tissues (e.g., heart tissue, prostate cancer samples) and negative controls

• Detection System:

  • For biotin-conjugated antibodies, use streptavidin-HRP or streptavidin-fluorophore conjugates

  • Signal amplification systems (e.g., TSA) can increase sensitivity but may amplify background

Quantification Methods:

• For IHC:

  • Use digital image analysis software (e.g., ImageJ with IHC plugins)

  • Quantify:

    • Staining intensity (0-3+ scale)

    • Percentage of positive cells

    • H-score (intensity × percentage, range 0-300)

  • Include multiple fields per sample (≥5)

• For Immunofluorescence:

  • Measure mean fluorescence intensity

  • Analyze subcellular distribution patterns

  • Colocalization with other markers can be quantified using Pearson's or Mander's coefficients

Data Analysis Best Practices:

• Score samples blindly to avoid bias

• Use standardized positive controls to normalize between batches

• For clinical samples, correlate with patient data (Gleason score, survival, etc.) using appropriate statistical methods

• Present data with appropriate statistical analysis (e.g., Kaplan-Meier curves for survival data)

• When comparing normal vs. disease tissues, use paired samples when possible

How can PDLIM5 antibodies be utilized in studying the protein's role in dendritic spine morphogenesis and neuronal function?

PDLIM5 contributes to dendritic spine morphogenesis and synaptic function, with specialized research approaches required to study these roles:

Experimental Approaches:

• Primary Neuronal Cultures:

  • Hippocampal or cortical neurons (DIV14-21) for mature spine formation

  • Transfection with PDLIM5 constructs (overexpression or knockdown)

  • Live imaging of spine dynamics using fluorescent markers

• Advanced Microscopy Techniques:

  • Super-resolution microscopy (STED, STORM) to visualize spine morphology changes

  • Multi-color imaging to localize PDLIM5 with synaptic markers (PSD-95, synaptophysin)

  • Time-lapse imaging to track spine morphology changes after stimulation

• Biochemical Analyses:

  • Synaptosomal fractionation to isolate postsynaptic densities

  • Co-immunoprecipitation to identify PDLIM5 binding partners in neurons

  • Proximity ligation assay to visualize protein interactions in situ

Functional Readouts:

• Morphological Analysis:

  • Spine density, length, head width, and classification (mushroom, thin, stubby)

  • Dendritic complexity (Sholl analysis)

  • Synapse formation rate and stability

• Electrophysiological Measurements:

  • Patch-clamp recordings to measure:

    • mEPSCs (miniature excitatory postsynaptic currents)

    • Paired-pulse facilitation

    • Long-term potentiation/depression

  • Multi-electrode arrays for network activity

Isoform-Specific Effects:

• Isoform 1 (but not isoform 2) expression favors spine thinning and elongation

• Design experiments to distinguish between isoform-specific effects using:

  • Isoform-specific antibodies

  • Selective overexpression of different isoforms

  • Rescue experiments with specific isoforms after knockdown

What advances in biotin-conjugated antibody technology are improving PDLIM5 detection sensitivity and specificity?

Recent technological advances have enhanced the utility of biotin-conjugated antibodies for PDLIM5 detection:

Enhanced Conjugation Chemistry:

• Site-specific biotinylation strategies that preserve antibody binding capacity

• Controlled biotin:antibody ratios to prevent over-biotinylation that can reduce affinity

• Use of longer spacer arms (e.g., biotin-XX) to improve accessibility to avidin/streptavidin

Novel Detection Systems:

• DSB-X Biotin Technology:

  • Enables reversible binding between biotin and avidin/streptavidin

  • Allows stripping and reprobing of the same sample

  • Particularly useful for sequential detection strategies

  • Can be used for isolating biotinylated complexes and then releasing them under mild conditions

• Signal Amplification Methods:

  • Tyramide signal amplification (TSA) combined with biotin-conjugated antibodies

  • Quantum dot-streptavidin conjugates for improved sensitivity and photostability

  • Rolling circle amplification (RCA) methods for extreme sensitivity

Reduction of Background Issues:

• Advanced blocking formulations specifically designed for biotin-based detection

• Pre-formed avidin-biotin complexes to reduce non-specific binding

• Addition of specific salts to buffers to minimize non-specific binding to nitrocellulose

Multiplexing Capabilities:

• Combination with other detection modalities (fluorescence, enzyme)

• Orthogonal biotin-binding systems with different specificities

• Sequential labeling and detection strategies enabled by DSB-X biotin technology

Application-Specific Optimizations:

• Flow cytometry: Optimized dilutions (1:100) with specific buffer formulations

• Western blot: Enhanced sensitivity through optimized blocking and washing steps

• ELISA: Improved coating strategies and detection limits

How can researchers integrate PDLIM5 expression data with clinical parameters for translational research applications?

Integrating PDLIM5 expression data with clinical parameters requires systematic approaches:

Data Integration Methodologies:

Clinical Correlation Protocols:

• Prospective Study Design:

  • Baseline PDLIM5 expression in patient samples

  • Longitudinal follow-up with defined endpoints

  • Integration with treatment response data

  • Standardized scoring systems for PDLIM5 expression

• Retrospective Analysis:

  • Match archived samples with comprehensive clinical datasets

  • Control for confounding variables (age, stage, treatment)

  • Use multivariate analysis to assess independent contribution of PDLIM5

Translational Applications:

• Biomarker Development:

  • Validate PDLIM5 as a prognostic marker in independent cohorts

  • Determine optimal cutoff values for "high" vs. "low" expression

  • Combine with existing biomarkers to improve predictive accuracy

• Therapeutic Targeting Assessment:

  • Correlate PDLIM5 expression with drug sensitivity

  • Evaluate PDLIM5 as a predictor of response to TGFβ pathway modulators

  • Develop companion diagnostics for targeted therapies

Standardization Recommendations:

• Use automated image analysis for consistent scoring

• Implement quality control measures for antibody lot consistency

• Normalize data against internal references

• Apply statistical methods appropriate for non-parametric biomarker data

What are the emerging research questions regarding PDLIM5's role in interactions between circRNAs and TGFβ signaling?

Recent discoveries regarding circZBTB46-PDLIM5-Smad interactions present several emerging research questions:

Mechanistic Questions:

• Regulatory Network Mapping:

  • How does circZBTB46 specifically recognize and bind PDLIM5?

  • What structural domains of PDLIM5 are involved in circRNA binding?

  • Do other circRNAs compete with circZBTB46 for PDLIM5 binding?

• Signaling Pathway Crosstalk:

  • How does TGFβ stimulation affect the circZBTB46-PDLIM5 interaction?

  • What intermediate factors mediate between TGFβ receptor activation and circZBTB46-PDLIM5 complex disruption?

  • Are there feedback mechanisms that restore circZBTB46-PDLIM5 complexes after prolonged TGFβ stimulation?

Methodological Approaches:

• Structure-Function Studies:

  • CRISPR-based domain mapping of PDLIM5 to identify circRNA binding regions

  • RNA structure probing to determine circZBTB46 secondary structures involved in binding

  • In vitro binding assays with purified components to establish direct interactions

• Advanced Imaging Techniques:

  • RNA-protein interaction visualization in living cells

  • FRET-based sensors to monitor complex formation/dissociation dynamics

  • Super-resolution microscopy to determine subcellular localization of complexes

• Systems Biology Approaches:

  • Network analysis of PDLIM5-interacting circRNAs across different tissues

  • Mathematical modeling of the circRNA-PDLIM5-Smad signaling network

  • Omics integration (RNA-seq, CLIP-seq, proteomics) to map global effects

Therapeutic Implications:

• Can circZBTB46-PDLIM5 interactions be targeted to modulate TGFβ signaling?

• Would disease-specific alterations in this pathway offer novel drug targets?

• Could circZBTB46 mimetics serve as TGFβ pathway modulators?

How might new biotinylation strategies improve the specificity and functionality of PDLIM5 antibodies in complex tissues?

Advancements in biotinylation chemistry offer promising approaches for next-generation PDLIM5 antibodies:

Novel Biotinylation Approaches:

• Site-Specific Biotinylation:

  • Enzymatic biotinylation using BirA ligase for precise conjugation

  • Incorporation of unnatural amino acids for click chemistry-based biotinylation

  • ScFv or Fab fragment biotinylation for improved tissue penetration

• Cleavable Linker Technologies:

  • Extension of DSB-X biotin technology with tissue-specific cleavable linkers

  • pH-sensitive linkages for endosomal escape

  • Photocleavable biotin derivatives for spatiotemporal control of detection

Application-Specific Improvements:

• For Complex Tissue Analysis:

  • Multi-epitope biotinylated antibodies targeting different PDLIM5 domains

  • Combination with tissue clearing technologies (CLARITY, iDISCO)

  • Integration with spatial transcriptomics for correlative analysis

• For Live Cell Imaging:

  • Membrane-permeable biotinylated antibody fragments

  • Two-step labeling strategies using biorthogonal chemistry

  • SNAP/CLIP-tag fusions for specific labeling of PDLIM5 isoforms

Detection System Advancements:

• Ultra-sensitive nanoprobes (quantum dots, upconversion nanoparticles)

• Multiplexed detection systems combining biotin with other tags

• Proximity-based amplification systems for single-molecule sensitivity

Clinical Translation Considerations:

• Reduced immunogenicity through deglycosylated streptavidin/NeutrAvidin systems

• Standardized production methods for consistent lot-to-lot performance

• Compatibility with automated staining platforms for clinical laboratory implementation