ZC3H14 Antibody, FITC conjugated

What is ZC3H14 protein and what are its primary functions in cellular processes?

ZC3H14 (Zinc Finger CCCH Domain-Containing Protein 14) belongs to a family of poly(A) binding proteins that regulate gene expression through influencing mRNA stability, nuclear export, and translation processes . It has been identified as essential for proper brain function, with mutations in the ZC3H14 gene linked to nonsyndromic, autosomal recessive intellectual disability . The protein contains CCCH-type zinc finger domains that enable RNA binding, particularly to polyadenosine sequences.

Research indicates ZC3H14 plays a critical role in proper poly(A) tail length maintenance, which affects RNA processing and metabolism . Mouse models lacking functional ZC3H14 (Zc3h14 Δex13/Δex13) demonstrate the importance of this protein in neurological functions . While ZC3H14 is ubiquitously expressed, its critical functions appear particularly important in neural tissues, making it an interesting subject for neurodevelopmental research.

What are the key considerations for validating the specificity of ZC3H14 antibody in experimental procedures?

When validating ZC3H14 antibody specificity, researchers should implement multiple complementary approaches:

• Knockout/knockdown validation: Compare antibody staining between wild-type and ZC3H14-deficient samples (using knockout models or siRNA knockdown). The Zc3h14 Δex13/Δex13 mouse model described in the literature can serve as an excellent negative control .

• Isoform awareness: ZC3H14 exists in multiple splice variants. The antibody should be validated against the specific isoform(s) of interest. Some antibodies may recognize a specific region present in certain isoforms but absent in others .

• Cross-reactivity testing: Test the antibody against known related proteins with similar structural domains to ensure specificity.

• Western blot analysis: Confirm the antibody detects bands of the expected molecular weight (the full-length ZC3H14 isoforms a-c are readily detectable in control samples) .

• Immunoprecipitation followed by mass spectrometry: This can confirm the identity of proteins recognized by the antibody, as demonstrated in studies where ZC3H14 was immunoprecipitated and analyzed .

Be aware that some truncated forms of ZC3H14 may be detected at lower molecular weights, as observed in some tissues where a small amount of a lower molecular weight band was detected by an N-terminal ZC3H14 antibody .

What is the recommended protocol for using ZC3H14 Antibody, FITC conjugated in immunofluorescence studies?

For optimal immunofluorescence results with FITC-conjugated ZC3H14 antibody:

Sample preparation:

• Culture cells on appropriate coverslips or slides

• Fix cells with 2% formaldehyde for 10 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 5 minutes

• Block with 5% normal serum (matching the secondary antibody host) in PBS with 0.1% BSA for 30-60 minutes

Antibody incubation:

• Dilute the FITC-conjugated ZC3H14 antibody at 1:1000 for fluorescence-based assays (adjust based on signal strength)

• Incubate samples with diluted antibody for 1-2 hours at room temperature or overnight at 4°C in a humidified chamber

• Wash 3 times with PBS to remove unbound antibody

• Counterstain nuclei with Hoechst to mark nuclear DNA

Visualization:

• Mount slides using anti-fade mounting medium

• Visualize using a fluorescence microscope with appropriate filter sets for FITC (excitation ~495 nm, emission ~519 nm)

• Images can be acquired using systems like the Olympus IX81 microscope with a 0.3 NA 100X objective

For co-localization studies, consider double staining with markers such as SC35 (for nuclear speckles), which can be detected using antibodies with different fluorophores such as Texas Red .

How can ZC3H14 Antibody, FITC conjugated be effectively utilized in studying RNA processing defects associated with neurological disorders?

ZC3H14's implication in intellectual disability makes its antibody valuable for investigating RNA processing defects in neurological disorders. A methodological approach includes:

In vitro neuronal culture systems:

• Establish primary neuron cultures or differentiated neural progenitor cells from control and disease models

• Apply the FITC-conjugated ZC3H14 antibody (dilution 1:100-500) to visualize localization patterns

• Compare subcellular distribution in healthy versus diseased states, with particular attention to nuclear/cytoplasmic ratios

RNA processing assessment:

• Combine ZC3H14 immunofluorescence with RNA FISH (fluorescence in situ hybridization) to visualize both the protein and its target RNAs

• Apply actinomycin D treatment (5 μg/ml) to inhibit transcription and monitor ZC3H14 relocalization

• Track poly(A) tail length changes using specialized techniques such as ePAT (extension poly(A) test) in ZC3H14-deficient versus normal cells

Stress response experiments:

• Subject neuronal cultures to various stressors (oxidative stress, heat shock)

• Monitor ZC3H14 localization changes using the FITC-conjugated antibody

• Correlate with RNA granule formation and translational control

Model system integration:
Compare findings between in vitro systems and mouse models such as the Zc3h14 Δex13/Δex13 mice, which show no detectable expression of ZC3H14 isoforms a-c . This approach allows validation across experimental systems and strengthens the translational relevance of findings.

What are the optimal parameters for quantitative analysis of ZC3H14 expression using FITC-conjugated antibodies in different neural cell types?

For rigorous quantitative analysis of ZC3H14 expression across neural cell types:

Sample standardization:

• Prepare consistent cell numbers (e.g., 1×10^5 cells) per sample

• Fix and permeabilize using standardized protocols (2% formaldehyde for 10 minutes followed by 0.1% Triton X-100 for 5 minutes)

• Process all samples in parallel to minimize technical variation

Antibody titration:
Perform a dilution series (1:100, 1:500, 1:1000, 1:5000) to determine the optimal concentration that provides specific signal with minimal background

Quantification methods:

• Flow cytometry: For population-level analysis

  • Generate single-cell suspensions from neural tissues or cultures

  • Stain with FITC-conjugated ZC3H14 antibody (1:1000 dilution)

  • Include unstained and isotype controls

  • Collect sufficient events (minimum 10,000 cells) per sample

  • Analyze median fluorescence intensity (MFI) across cell populations

• Microscopy-based quantification:

  • Acquire Z-stack images maintaining consistent exposure settings

  • Employ automated image analysis software to segment cells and quantify fluorescence intensity

  • Measure nuclear vs. cytoplasmic signal independently

  • Normalize to cell area or volume and appropriate reference markers

Cell type-specific considerations:

How can researchers differentiate between specific ZC3H14 isoforms using the FITC-conjugated antibody in experimental contexts?

Differentiating between ZC3H14 isoforms requires strategic approaches:

Epitope mapping:
First, determine which epitope the FITC-conjugated antibody recognizes. The antibodies available target different regions:

• Some target AA 176-306

• Others target internal regions

• Some specifically recognize the N-terminal domain (first 97 amino acids of isoform 1)

Understanding the specific binding region is crucial for isoform discrimination.

Isoform-specific experimental design:

• Western blot analysis with size discrimination:

  • Use the unconjugated version of the same antibody clone in Western blots

  • Different isoforms will appear as distinct bands at different molecular weights

  • Compare observed bands with expected molecular weights of known isoforms

• Immunoprecipitation coupled with specific detection:

  • Use the antibody to immunoprecipitate ZC3H14

  • Analyze precipitated proteins by mass spectrometry to identify specific isoforms

  • Look for peptides that map to isoform-specific regions

• Combined immunofluorescence approaches:

  • Use the FITC-conjugated ZC3H14 antibody alongside antibodies against proteins known to interact specifically with certain isoforms

  • Different subcellular localization patterns may indicate different isoforms

• Validation with genetic models:

  • Utilize samples from the Zc3h14 Δex13/Δex13 mouse model, which lacks expression of isoforms a-c

  • This provides a controlled system to validate isoform specificity

Technical considerations:

• When interpreting results, be aware that truncated forms may be detected, as observed in some mouse tissues where a lower molecular weight band was detected by an N-terminal ZC3H14 antibody

• Quantify relative isoform expression using densitometry analysis when performing Western blots

• Always include appropriate positive and negative controls specific to each isoform

What are the most effective experimental designs for studying ZC3H14 interactions with target RNAs using FITC-conjugated antibodies?

To investigate ZC3H14 interactions with target RNAs using FITC-conjugated antibodies, consider these approaches:

RNA-protein co-localization:

• Combined IF-FISH technique:

  • Perform immunofluorescence with FITC-conjugated ZC3H14 antibody

  • Follow with fluorescence in situ hybridization (FISH) using probes targeting suspected RNA targets

  • Use spectrally distinct fluorophores (e.g., Cy3 or Texas Red for RNA detection)

  • Analyze co-localization using confocal microscopy and quantitative co-localization metrics

• Proximity ligation assay (PLA)-based detection:

  • Combine ZC3H14 antibody with antibodies against RNA modifications (e.g., m6A)

  • PLA signal indicates close proximity between ZC3H14 and modified RNAs

Dynamic interaction analysis:

• FRAP (Fluorescence Recovery After Photobleaching):

  • Transfect cells with fluorescently tagged RNA constructs

  • Use FITC-conjugated ZC3H14 antibody in fixed cells at different timepoints

  • Measure dynamics of interaction following stress or other perturbations

• Live-cell RNA tracking with fixed-cell ZC3H14 detection:

  • Track labeled RNAs in living cells

  • Fix at specific timepoints

  • Detect ZC3H14 using the FITC-conjugated antibody

  • Correlate RNA movements with ZC3H14 localization

Functional validation approaches:

• CLIP-seq correlation:

  • Perform CLIP-seq (Cross-linking immunoprecipitation sequencing) to identify RNA targets of ZC3H14

  • Use FITC-conjugated antibody to visualize cellular distribution of ZC3H14

  • Correlate spatial distribution with identified RNA targets

• RNA stability assessment:

  • Treat cells with transcription inhibitors (actinomycin D, 5 μg/ml)

  • Track ZC3H14-RNA co-localization over time using FITC-antibody and RNA FISH

  • Correlate with RNA half-life measurements

Experimental controls:

• Include RNase treatment controls to confirm RNA-dependency of interactions

• Use cells from Zc3h14 Δex13/Δex13 mice as negative controls

• Include competition experiments with unlabeled antibody to confirm specificity

What are the current limitations and future directions for ZC3H14 Antibody, FITC conjugated applications in molecular neuroscience?

Current limitations:

• Isoform specificity challenges:

  • Most available antibodies cannot unambiguously distinguish between all ZC3H14 isoforms

  • Lower molecular weight isoforms or truncated forms may be difficult to characterize precisely

• Temporal resolution limitations:

  • Fixed-cell immunofluorescence provides only snapshots of ZC3H14 localization

  • Dynamic protein-RNA interactions are challenging to capture with antibody-based approaches

• Penetration in tissue samples:

  • FITC-conjugated antibodies may have limited penetration in thick tissue sections

  • Signal-to-noise ratio can be problematic in complex neural tissues

• Quantification standardization:

  • Lack of standardized quantification methods across studies hinders comparative analysis

  • Fluorescence intensity can vary between batches and experimental conditions

Future directions and methodological advances:

• Super-resolution microscopy applications:

  • Implement STED, STORM or PALM microscopy to precisely localize ZC3H14 in relation to RNA granules

  • Combine with expansion microscopy for improved spatial resolution in dense neural tissues

• Multi-omics integration:

  • Correlate immunofluorescence data with RNA-seq, CLIP-seq, and proteomics

  • Develop computational frameworks to integrate spatial and -omics data

• In vivo applications:

  • Adapt techniques for in vivo imaging in model organisms

  • Develop clearing protocols compatible with FITC-conjugated antibodies for whole-brain imaging

• Functional characterization:

  • Combine FITC-ZC3H14 visualization with electrophysiological recordings in neurons

  • Correlate ZC3H14 localization patterns with functional outcomes

• Single-cell approaches:

  • Implement single-cell analysis techniques to capture cell-to-cell variability in ZC3H14 expression and localization

  • Develop microfluidic approaches for high-throughput analysis