PDLIM5 Antibody, FITC conjugated

What is PDLIM5 and why is it relevant to research?

PDLIM5 is a scaffold protein member of the PDZ-LIM protein family that participates in signal-transducing regulation of membrane-associated proteins, cytoskeletal proteins, and various signaling molecules. It has been implicated in tumor progression, drug resistance mechanisms, and neuropsychiatric disorders. PDLIM5 is overexpressed in non-small cell lung cancer (NSCLC) and associated with poor prognosis. It has also been genetically linked to mood disorders, with upregulated expression in postmortem brains of patients with bipolar disorder and downregulated expression in peripheral lymphocytes of patients with major depression .

What are the key structural features of PDLIM5?

PDLIM5 contains a PDZ domain and three LIM domains. The PDZ domain is encoded partly by exon 2, while exon 9 specifies the beginning of the first LIM domain. These structural features are critical because the LIM domains mediate protein-protein interactions, particularly with protein kinase C (PKC). Studies with knockout models have demonstrated that the LIM domain is essential for embryonic development, as homozygous knockout of Pdlim5 is embryonic lethal .

What applications is the PDLIM5 Antibody, FITC conjugated suitable for?

The PDLIM5 Antibody, FITC conjugated has been tested and validated for ELISA applications. The antibody is a polyclonal IgG derived from rabbit, using recombinant Human PDZ and LIM domain protein 5 protein (268-404AA) as the immunogen. Its FITC conjugation makes it particularly valuable for direct fluorescent detection methods without requiring secondary antibodies .

What are the proper storage conditions for PDLIM5 Antibody, FITC conjugated?

The antibody should be stored at -20°C or -80°C upon receipt. It's critical to avoid repeated freeze-thaw cycles as these can compromise antibody activity and binding specificity. The antibody is supplied in liquid form in a buffer containing 0.03% Proclin 300 as a preservative and 50% Glycerol in 0.01M PBS, pH 7.4 .

How should I design experiments to study PDLIM5 signaling pathways?

When designing experiments to study PDLIM5 signaling pathways, consider its role in the AMPK/TSC2/mTORC1 pathway, particularly in cancer research contexts. PDLIM5 directly binds to AMPK, maintaining its activation and inhibiting degradation. Experimental approaches should include:

• Knockdown experiments using siRNA targeting PDLIM5 (consider using multiple siRNA sequences as shown in the literature)

• Protein interaction studies (co-immunoprecipitation) to verify binding partners

• Phosphorylation state analysis of downstream targets in the pathway (TSC2, mTOR)

• Cell viability and apoptosis assays following PDLIM5 modulation

• Drug resistance studies particularly in NSCLC models

What controls should I include when using PDLIM5 Antibody, FITC conjugated?

For rigorous experimental design with PDLIM5 Antibody, FITC conjugated, include:

• Negative controls:

  • Isotype control (rabbit IgG, FITC conjugated)

  • Secondary antibody-only control (where applicable)

  • Unstained samples for autofluorescence baseline

• Positive controls:

  • Tissues/cells known to express PDLIM5 (heart, brain, skeletal muscle)

  • Recombinant PDLIM5 protein

• Knockdown validation:

  • Samples treated with verified PDLIM5 siRNA to demonstrate specificity

How can I validate PDLIM5 knockdown efficiency when using siRNA approaches?

To validate PDLIM5 knockdown efficiency:

• Design multiple siRNA sequences targeting different regions of PDLIM5 mRNA (refer to table below for validated sequences)

• Use quantitative RT-PCR to measure PDLIM5 mRNA levels

• Employ Western blot with a validated PDLIM5 antibody to assess protein reduction

• Include a scrambled siRNA control (e.g., FITC-SCR siRNA as shown in the literature)

• Consider using fluorescently labeled siRNA for transfection efficiency monitoring

• Optimize transfection conditions based on cell type

How can I use PDLIM5 Antibody, FITC conjugated in cancer research protocols?

For cancer research applications focusing on PDLIM5:

• Expression Analysis in NSCLC Tissues:

  • Use PDLIM5 Antibody, FITC conjugated for direct immunofluorescence staining of tissue sections

  • Correlate expression levels with clinical outcomes and drug resistance patterns

  • Combine with markers of AMPK pathway activation

• Drug Resistance Mechanisms:

  • Monitor PDLIM5 expression changes during development of EGFR-TKI resistance

  • Combine with gefitinib treatment protocols in resistant vs. sensitive cell lines

  • Use flow cytometry with FITC-conjugated antibody to quantify expression changes at single-cell level

• Targeted Delivery Systems:

  • Incorporate PDLIM5 siRNA into nanobubble delivery systems

  • Use ultrasound-mediated delivery to enhance transfection efficiency

  • Monitor both knockdown efficiency and phenotypic consequences on cell proliferation, migration, and apoptosis

What considerations are important when studying PDLIM5 in neuropsychiatric disorders?

When investigating PDLIM5 in neuropsychiatric contexts:

• Expression Pattern Analysis:

  • Compare PDLIM5 expression across brain regions using the FITC-conjugated antibody

  • Correlate with psychiatric phenotypes (bipolar disorder, depression)

• Pharmacological Interventions:

  • Monitor PDLIM5 expression changes in response to psychiatric medications

  • Note that chronic imipramine treatment increases Pdlim5 mRNA in the hippocampus

  • Consider that methamphetamine increases while haloperidol decreases Pdlim5 mRNA in the prefrontal cortex

• Behavioral Correlates:

  • Utilize animal models with reduced Pdlim5 expression

  • Assess behaviors relevant to mood disorders (forced swimming test, locomotor activity)

  • Consider that Pdlim5 heterozygous knockout mice show increased immobility in forced swimming tests, suggesting depression-like behavior

How can I employ PDLIM5 Antibody, FITC conjugated in multiplex immunofluorescence protocols?

For advanced multiplex immunofluorescence:

• Spectral Compatibility:

  • FITC has excitation/emission peaks at approximately 495/519 nm

  • Pair with fluorophores in distinct spectral ranges (e.g., Cy3, Cy5, APC)

  • Employ sequential scanning in confocal microscopy to minimize bleed-through

• Co-localization Studies:

  • Combine with antibodies against interaction partners (PKC, AMPK)

  • Use Z-stack imaging to confirm spatial relationships in three dimensions

  • Employ quantitative co-localization analysis (Pearson's or Mander's coefficients)

• Live Cell Applications:

  • For tracking dynamics, confirm antibody internalization does not alter function

  • Validate that FITC photobleaching rates allow adequate imaging time

  • Consider photobleaching correction methods for quantitative applications

What are common pitfalls when using PDLIM5 Antibody, FITC conjugated and how can they be addressed?

Common challenges and solutions:

• High Background Signal:

  • Increase blocking time/concentration (BSA or serum)

  • Reduce primary antibody concentration

  • Include additional washing steps

  • Use appropriate negative controls to identify source of background

• Weak or Absent Signal:

  • Confirm PDLIM5 expression in your sample type

  • Optimize antibody concentration through titration

  • Consider antigen retrieval methods for fixed samples

  • Verify storage conditions have not compromised antibody activity

• Non-specific Binding:

  • Pre-absorb antibody with control protein

  • Increase stringency of wash buffers

  • Validate signal with orthogonal methods (Western blot, qPCR)

  • Compare with non-conjugated PDLIM5 antibody results

How can I optimize PDLIM5 siRNA delivery for maximum knockdown efficiency?

To optimize PDLIM5 siRNA delivery:

• Delivery Method Selection:

  • For standard cell culture: Compare lipid-based transfection reagents

  • For difficult-to-transfect cells: Consider electroporation or viral vectors

  • For in vivo applications: Explore nanobubble carriers with ultrasound-mediated delivery

• Protocol Optimization:

  • Titrate siRNA concentration (typically 10-50 nM)

  • Adjust cell density at transfection time

  • Optimize transfection reagent-to-siRNA ratio

  • Determine optimal post-transfection timing for experiments

• Validation Approaches:

  • Use FITC-labeled control siRNA to monitor uptake efficiency

  • Employ laser scanning confocal microscopy to confirm cellular distribution

  • Quantify knockdown at both mRNA (qRT-PCR) and protein (Western blot) levels

How should I interpret changes in PDLIM5 expression in different experimental contexts?

When interpreting PDLIM5 expression changes:

• Cancer Research Context:

  • Increased PDLIM5 expression may indicate poor prognosis in NSCLC

  • Changes may correlate with drug resistance mechanisms via AMPK/TSC2/mTORC1 pathway

  • Reduction in PDLIM5 may inhibit tumor cell proliferation and promote apoptosis

• Neuropsychiatric Research Context:

  • Interpret based on brain region (prefrontal cortex vs. hippocampus)

  • Consider opposing expression patterns in bipolar disorder (increased) vs. major depression (decreased)

  • Take into account effects of psychotropic medications (imipramine increases, haloperidol decreases expression)

• Developmental Biology Context:

  • Complete loss of PDLIM5 is embryonically lethal

  • Partial reduction may affect cardiac development through PKC scaffolding functions

  • May influence muscle satellite cell proliferation and differentiation via p38-MAPK pathway

What statistical approaches are appropriate for analyzing PDLIM5 expression data across experimental groups?

For rigorous statistical analysis:

• For Comparing Expression Levels:

  • Use t-tests for two-group comparisons (with appropriate tests for normality)

  • Apply ANOVA with post-hoc tests for multiple group comparisons

  • Consider non-parametric alternatives if normality assumptions are violated

• For Correlation Analysis:

  • Apply Pearson's or Spearman's correlation to relate PDLIM5 levels to clinical outcomes

  • Use regression models to account for covariates

  • Consider survival analysis (Kaplan-Meier, Cox regression) for prognostic studies

• For High-dimensional Data:

  • When combining with transcriptomic data, apply appropriate multiple testing corrections

  • Consider dimension reduction techniques for complex datasets

  • Validate findings with independent cohorts or orthogonal methods

How can I differentiate between the roles of different PDLIM5 isoforms in my experimental system?

To distinguish between PDLIM5 isoforms:

• Isoform Identification:

  • The gene-trap vector described in the literature generates a fusion between the PDZ domain and β-geo, containing 1–368 amino acids of the ENH1 isoform and 1–306 aa of the ENH2 isoform

  • Use isoform-specific primers for RT-PCR analysis

  • Consider Western blotting with antibodies recognizing different domains

• Functional Analysis:

  • Design isoform-specific siRNAs where possible

  • Use domain-specific constructs for overexpression studies

  • Consider the differential interactions of isoforms with binding partners

• Data Interpretation:

  • Recognize that the PDLIM5 Antibody, FITC conjugated may not distinguish between all isoforms

  • Correlate isoform-specific expression with particular phenotypes

  • Note that some functions may be isoform-specific while others are shared

How might PDLIM5 targeting be combined with other therapeutic approaches in cancer research?

Emerging combinatorial approaches include:

• With EGFR-TKI Therapy:

  • PDLIM5 knockdown may enhance sensitivity to gefitinib and other EGFR-TKIs

  • Combined targeting could overcome resistance mechanisms in NSCLC

  • Consider sequential vs. simultaneous treatment protocols

• With Metabolic Pathway Inhibitors:

  • PDLIM5 affects AMPK/mTOR signaling, suggesting synergy with mTOR inhibitors

  • Explore combinations with compounds affecting cellular energy metabolism

  • Monitor both tumor growth inhibition and potential systemic side effects

• With Nanobubble-Based Delivery Systems:

  • Ultrasound-sensitive siRNA-loaded nanobubbles show promise for PDLIM5 targeting

  • This approach allows targeted delivery to tumor sites

  • Could be combined with conventional chemotherapeutics for enhanced efficacy

What techniques are emerging for studying PDLIM5 protein interactions in living systems?

Advanced methodologies include:

• Proximity Labeling Approaches:

  • BioID or APEX2 fusion proteins to identify proximal interactors of PDLIM5

  • TurboID for faster labeling kinetics in dynamic systems

  • Spatially restricted enzymatic tagging to identify compartment-specific interactions

• Live Cell Imaging Techniques:

  • FRET-based biosensors to monitor PDLIM5 interactions in real time

  • Split fluorescent protein complementation to visualize protein-protein associations

  • Optogenetic approaches to induce or disrupt interactions with temporal control

• In vivo Models:

  • Consider heterozygous Pdlim5 knockout mice for behavioral and biochemical studies

  • Tissue-specific conditional knockout models to overcome embryonic lethality

  • CRISPR-mediated tagging for endogenous protein tracking