DICER1 Antibody, HRP conjugated

What is DICER1 and why is it important in research applications?

DICER1 is a member of the RNase III family that specifically cleaves double-stranded RNAs to generate microRNAs (miRNAs). The protein plays a central role in short dsRNA-mediated post-transcriptional gene silencing. After long primary transcript pri-miRNAs are processed to stem-looped pre-miRNAs by Drosha, pre-miRNAs are transported to the cytoplasm and further processed by DICER1 to produce 22-nucleotide mature miRNAs. The mature miRNA then becomes part of the RNA-Induced Silencing Complex (RISC) and can bind to the 3' UTR of target mRNAs, regulating their expression . DICER1 functions as a double-stranded RNA endoribonuclease that cleaves naturally occurring long dsRNAs and short hairpin pre-microRNAs into fragments of twenty-one to twenty-three nucleotides with 3' overhang of two nucleotides, producing short interfering RNAs (siRNA) and mature microRNAs respectively . Because of its central role in gene regulation and potential involvement in disease processes, DICER1 is an important research target across multiple fields including developmental biology, cancer research, and genetics.

What types of HRP-conjugated DICER1 antibodies are available for research?

Based on current data, there are several types of HRP-conjugated DICER1 antibodies available for research purposes:

• Mouse monoclonal antibodies:

  • Targeting amino acids 1638-1899 of mouse DICER1 (clone S167-7, IgG1 isotype)

  • Targeting amino acids 1666-1922 of human DICER1 (clone OTI2B1, IgG2a isotype)

• Rabbit polyclonal antibodies:

  • Broad epitope recognition with reactivity to human, mouse, and rat DICER1

These antibodies differ in their host species, clonality, epitope recognition, and optimal applications, allowing researchers to select the most appropriate reagent for their specific experimental needs.

What are the recommended applications and dilutions for HRP-conjugated DICER1 antibodies?

HRP-conjugated DICER1 antibodies have been validated for multiple applications with specific recommended dilutions:

It is important to note that these are recommended starting dilutions, and optimization may be necessary for specific experimental conditions. The optimal dilutions should be determined by the user through titration experiments for their particular application and sample type .

What species reactivity do HRP-conjugated DICER1 antibodies demonstrate?

The available HRP-conjugated DICER1 antibodies show cross-reactivity across multiple species:

• Mouse monoclonal antibody (AA 1638-1899): Cross-reactive with human, mouse, and rat DICER1

• Rabbit polyclonal antibody: Reactive with human, mouse, and rat DICER1

• Mouse monoclonal antibody (Clone OTI2B1): Reactive with human, mouse, and rat DICER1

How should Western blot protocols be optimized when using HRP-conjugated DICER1 antibodies?

When optimizing Western blot protocols for HRP-conjugated DICER1 antibodies, several considerations are important:

• Protein size detection: DICER1 is a large protein (~215 kDa) , requiring special attention to transfer efficiency. Extended transfer times or specialized transfer methods for high molecular weight proteins may be necessary.

• Sample preparation: Complete lysis of nuclear components is essential since DICER1 can localize to both cytoplasmic and nuclear compartments, as evidenced by immunofluorescence studies .

• Antibody dilution: Start with the recommended dilution (typically 1:1000 for Western blotting) and adjust based on signal-to-noise ratio.

• Blocking optimization: Since HRP-conjugated antibodies eliminate the need for secondary antibodies, blocking conditions become more critical to reduce background. PBS with 3-5% non-fat dry milk or BSA is typically effective.

• Detection substrate selection: For optimal sensitivity, choose an enhanced chemiluminescence (ECL) substrate appropriate for the expected expression level of DICER1 in your samples.

• Positive controls: Include a positive control such as cell lines known to express DICER1 (e.g., TPC1 cells as mentioned in research on thyroid carcinoma) .

• Loading controls: Due to DICER1's size, stripping and reprobing membranes can be challenging, so consider loading duplicate gels for probing with housekeeping protein antibodies.

What methodological considerations are important for immunohistochemistry with HRP-conjugated DICER1 antibodies?

For successful immunohistochemistry (IHC) using HRP-conjugated DICER1 antibodies, researchers should consider:

• Fixation and antigen retrieval: DICER1 epitopes may be sensitive to overfixation. Standard formalin fixation (10% neutral buffered formalin) is appropriate , but optimization of antigen retrieval methods is essential for optimal staining.

• Dilution optimization: Begin with recommended dilutions (1:100-500 for IHC-P with polyclonal antibodies or 1:150 for monoclonal antibodies ) and adjust as needed.

• Background reduction: Since the antibody is HRP-conjugated, endogenous peroxidase activity must be thoroughly quenched (typically using 3% hydrogen peroxide in methanol) before antibody application.

• Incubation conditions: Longer incubation times at 4°C may improve specific binding while reducing background.

• Controls: Include positive tissue controls (tissues known to express DICER1) and negative controls (omitting primary antibody) to validate staining specificity.

• Counterstaining: Light hematoxylin counterstaining helps visualize tissue architecture without obscuring specific DICER1 staining.

• DICER1 localization consideration: DICER1 can show both cytoplasmic and nuclear localization depending on the cell type and physiological state, as evidenced in testicular tissue studies . Evaluate staining patterns carefully in this context.

How can researchers effectively use HRP-conjugated DICER1 antibodies in immunofluorescence applications?

For immunofluorescence (IF) applications with HRP-conjugated DICER1 antibodies:

• Signal amplification: Although HRP is primarily used for colorimetric detection, HRP-conjugated antibodies can be used in IF through tyramide signal amplification (TSA) methods. This approach converts the HRP enzymatic activity into a fluorescent signal.

• Dilution optimization: Begin with the recommended dilution of 1:100 for IF applications and adjust based on signal strength and background.

• Dual labeling considerations: When performing multi-label IF, perform the DICER1 detection first using the TSA method, then heat-inactivate the HRP before proceeding with other antibodies to prevent cross-reactivity.

• Subcellular localization analysis: Pay particular attention to the subcellular distribution of DICER1, which can vary depending on cell type and physiological state. Studies have documented both cytoplasmic and nuclear localization patterns .

• Fixation methods: Cross-linking fixatives like paraformaldehyde (4%) typically preserve DICER1 epitopes well for IF applications.

• Confocal microscopy: Due to DICER1's involvement in specific subcellular compartments related to RNA processing, confocal microscopy is recommended for detailed localization studies.

How do monoclonal and polyclonal HRP-conjugated DICER1 antibodies differ in research applications?

The choice between monoclonal and polyclonal HRP-conjugated DICER1 antibodies depends on specific research needs:

Monoclonal antibodies (e.g., mouse anti-DICER1, clone S167-7 or OTI2B1):

• Provide consistent lot-to-lot reproducibility due to recognition of a single epitope

• Offer higher specificity but potentially lower sensitivity

• Particularly useful for applications requiring precise epitope targeting, such as studying specific domains of DICER1 (e.g., amino acids 1638-1899) or (1666-1922)

• Ideal for quantitative studies where batch consistency is critical

• May be less tolerant to variations in sample preparation methods

Polyclonal antibodies (e.g., rabbit anti-DICER1):

• Recognize multiple epitopes on the DICER1 protein

• Generally provide higher sensitivity but potentially more background

• Better tolerance to protein denaturation and fixation conditions

• Particularly useful for detecting low-abundance DICER1 expression

• Valuable for applications where maximum signal is prioritized over epitope specificity

• May show higher batch-to-batch variation

For specific research questions, such as studying DICER1 phosphorylation states or investigating how mutations affect specific domains, monoclonal antibodies targeting particular regions would be preferred. For general detection of DICER1 expression in varied sample types, polyclonal antibodies might provide more robust results.

What approaches are recommended for studying DICER1 phosphorylation using HRP-conjugated antibodies?

DICER1 phosphorylation research requires specific methodological considerations:

• Combined antibody approach: While HRP-conjugated antibodies are valuable for detection, studying phosphorylation often requires using both total DICER1 antibodies and phospho-specific antibodies. Research on DICER1 phosphorylation has utilized both total Dicer1 antibody and phospho-Dicer-specific antibody in parallel .

• Phosphatase inhibitors: Sample preparation must include robust phosphatase inhibitor cocktails to preserve phosphorylation states.

• Validation of phosphorylation: Validation can be performed by comparing samples treated with and without phosphatase to confirm specificity of phospho-antibody detection.

• Subcellular localization analysis: Phosphorylation can affect DICER1 localization. Research has shown that constitutive Dicer1 phosphorylation (at sites S1712 and S1836) can influence its cellular distribution and function .

• Functional correlations: When studying phosphorylation, correlate antibody detection with functional assays measuring DICER1 activity, such as miRNA processing efficiency.

• Model systems: Consider using phosphomimetic mutants (e.g., S1712D and S1836D) as positive controls for phosphorylation studies, similar to those used in mouse models to study accelerated metabolism and aging .

• Quantification methodology: For accurate quantification of phosphorylated versus total DICER1, use digital image analysis with appropriate normalization controls.

How can researchers troubleshoot non-specific binding when using HRP-conjugated DICER1 antibodies?

When encountering non-specific binding with HRP-conjugated DICER1 antibodies, consider these troubleshooting steps:

• Optimize blocking conditions: Increase blocking time or concentration of blocking agent (BSA or non-fat dry milk). For particularly problematic samples, consider alternative blocking agents like casein or commercial blocking solutions.

• Titrate antibody concentration: Perform a dilution series beyond the recommended range to identify the optimal concentration that maximizes specific signal while minimizing background.

• Modify washing procedures: Increase the number and duration of wash steps, particularly after antibody incubation. Consider adding low concentrations of detergent (0.05-0.1% Tween-20) to wash buffers.

• Pre-absorb the antibody: For tissues with high endogenous biotin or other sources of non-specific binding, pre-absorption of the antibody with tissue lysate from a negative control sample can reduce background.

• Evaluate fixation impact: Overfixation can create artifactual binding sites. Test different fixation protocols if possible.

• Consider temperature effects: Performing antibody incubation at 4°C rather than room temperature often reduces non-specific interactions.

• Validate with alternative detection: If possible, compare results from HRP-conjugated antibodies with unconjugated primary antibodies and separate secondary detection to identify if the conjugation is contributing to background issues.

• Tissue-specific considerations: For certain tissues, such as thyroid tissue when studying DICER1's role in thyroid cancer , specific pre-treatments may be necessary to reduce endogenous background.

What methodological approaches can detect DICER1 in different subcellular compartments?

DICER1 can localize to different subcellular compartments depending on cell type and physiological state, requiring specialized approaches for comprehensive detection:

• Subcellular fractionation: Perform biochemical fractionation to separate nuclear, cytoplasmic, and membrane compartments before Western blotting with HRP-conjugated DICER1 antibodies.

• Confocal microscopy for co-localization: Use immunofluorescence with HRP-conjugated DICER1 antibodies (via TSA amplification) and co-stain with markers for specific subcellular structures:

  • Nuclear envelope (Lamin B)

  • Endoplasmic reticulum (Calnexin)

  • P-bodies (DCP1a)

  • Stress granules (G3BP1)

• Quantitative assessment: For nuclear localization, quantification methods similar to those described in testicular tissue studies can be employed, where cells with >50% nuclear DICER1 staining were counted as positive for nuclear localization .

• Super-resolution microscopy: For detailed localization studies, techniques like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) provide higher resolution than conventional confocal microscopy.

• Live-cell imaging: While HRP-conjugated antibodies are not suitable for live-cell studies, complementary approaches using fluorescent protein-tagged DICER1 can provide dynamic localization information to correlate with fixed-cell antibody studies.

• Electron microscopy: For ultrastructural localization, immunogold labeling with DICER1 antibodies can precisely map DICER1 to specific subcellular structures.

How can HRP-conjugated DICER1 antibodies be used in cancer research?

DICER1 has important implications in cancer biology, with specific methodological approaches for cancer research:

• Expression analysis in tumor samples: HRP-conjugated DICER1 antibodies can be used in IHC to evaluate DICER1 expression across tumor types and correlate with clinical outcomes. Studies have shown that DICER1 mRNA expression is downregulated in papillary thyroid carcinoma (PTC) .

• Tumor microarray studies: Standardized IHC protocols using HRP-conjugated DICER1 antibodies (dilution 1:100-500) enable high-throughput screening of DICER1 expression across multiple tumor samples simultaneously.

• Cell line models: Western blotting with HRP-conjugated DICER1 antibodies can quantify DICER1 expression in cancer cell lines, such as TPC1 thyroid cancer cells . This approach can help establish relationships between DICER1 levels and cancer cell behaviors.

• DICER1 haploinsufficiency models: Heterozygous DICER1 (+/-) cell lines created using CRISPR-Cas9 technology can be analyzed with HRP-conjugated DICER1 antibodies to understand dosage-dependent functions, as demonstrated in thyroid cancer research .

• DICER1 syndrome research: For investigations related to DICER1 syndrome (a rare genetic disorder predisposing to various tumors) , HRP-conjugated DICER1 antibodies can help characterize protein expression in patient-derived samples.

• miRNA processing analysis: Since DICER1 is crucial for miRNA biogenesis, cancer studies often examine relationships between DICER1 expression (detected via HRP-conjugated antibodies) and alterations in miRNA profiles that contribute to oncogenesis.

What considerations are important when investigating DICER1 in genetic disorders?

When studying DICER1 in genetic disorders like DICER1 syndrome:

• Mutation-specific protein detection: HRP-conjugated DICER1 antibodies can help assess how specific mutations affect protein expression, stability, and localization. This is particularly relevant for DICER1 syndrome, where germline pathogenic variants predispose to tumor development .

• Tissue-specific expression analysis: Since DICER1 syndrome affects multiple organs, IHC with HRP-conjugated DICER1 antibodies can characterize tissue-specific expression patterns in affected and unaffected tissues.

• Pathogenic variant impact: For known pathogenic DICER1 variants, compare protein expression and localization patterns between wild-type and mutant samples using standardized IHC or Western blot protocols with HRP-conjugated antibodies.

• Genotype-phenotype correlations: Correlate DICER1 protein expression patterns (detected with HRP-conjugated antibodies) with specific mutations and clinical manifestations in patient samples.

• Surveillance protocol development: DICER1 protein expression data can inform surveillance protocols for DICER1 syndrome carriers. Current research suggests that germline DICER1 pathogenic variants may be more common in the general population than previously thought, with an incidence of approximately 1:5121 based on gnomAD data .

• Functional domain considerations: Select HRP-conjugated DICER1 antibodies that target specific functional domains relevant to the mutations being studied. For example, antibodies targeting amino acids 1638-1899 versus 1666-1922 may provide different insights depending on the mutation location.

What are the essential controls for validating HRP-conjugated DICER1 antibody specificity?

Proper validation of HRP-conjugated DICER1 antibodies requires several controls:

• Positive controls: Include samples known to express DICER1, such as:

  • Well-characterized cell lines with documented DICER1 expression

  • Tissues with established DICER1 expression patterns

  • Recombinant DICER1 protein (for Western blot)

• Negative controls:

  • DICER1 knockout or knockdown samples (e.g., using CRISPR-Cas9 or siRNA technology)

  • Samples processed identically but with omission of primary antibody

  • Isotype controls matching the antibody class (IgG1 or IgG2a for monoclonal antibodies)

• Specificity validation:

  • Peptide competition assays using the immunizing peptide (e.g., amino acids 1638-1899 or 1666-1922 )

  • Detection of expected molecular weight (~215 kDa) in Western blot

  • Comparison of staining patterns with multiple DICER1 antibodies targeting different epitopes

• Sensitivity assessment:

  • Titration series with decreasing amounts of DICER1 protein

  • Comparison with established DICER1 antibodies of known sensitivity

• Cross-reactivity testing:

  • Evaluate specificity across species when working with human, mouse, and rat samples

  • Assess potential cross-reactivity with related RNase III family members

How should researchers approach reproducibility challenges with DICER1 antibodies in different experimental systems?

Addressing reproducibility challenges requires systematic approaches:

• Standardized protocols: Develop detailed standard operating procedures (SOPs) for each application (WB, IHC, IF) with HRP-conjugated DICER1 antibodies, documenting all variables:

  • Sample preparation methods

  • Buffer compositions

  • Incubation times and temperatures

  • Detection systems

• Antibody information documentation:

  • Record complete antibody information including catalog number, lot number, and dilution

  • For monoclonal antibodies, document the clone (e.g., S167-7 or OTI2B1 )

  • For polyclonal antibodies, record the host species and immunization strategy

• Validation across systems:

  • When transitioning between experimental systems (e.g., cell lines to tissue samples), perform parallel validations

  • Establish positive controls specific to each experimental system

• Inter-laboratory validation:

  • Exchange protocols and samples with collaborating laboratories

  • Perform blind analysis of identical samples across different labs

• Quantification methods:

  • Implement standardized quantification approaches for DICER1 expression

  • Use digital image analysis with defined parameters for IHC/IF

  • Include calibration standards for Western blot quantification

• Reporting guidelines:

  • Follow minimum information about antibody guidelines in publications

  • Deposit detailed protocols in repositories like protocols.io

This systematic approach helps ensure that findings with HRP-conjugated DICER1 antibodies are robust and reproducible across different experimental contexts.

How can HRP-conjugated DICER1 antibodies be integrated into multi-omics research approaches?

Integrating HRP-conjugated DICER1 antibodies into multi-omics research requires coordinated experimental design:

• Proteomics integration:

  • Use HRP-conjugated DICER1 antibodies for validation of mass spectrometry findings

  • Apply to immunoprecipitation followed by proteomics to identify DICER1 interaction partners

  • Correlate DICER1 protein levels (detected by antibodies) with global proteome changes

• Transcriptomics correlation:

  • Combine DICER1 protein detection with RNA sequencing data to correlate protein expression with transcriptional effects, as demonstrated in studies showing transcriptomic alterations when DICER1 is depleted

  • Use DICER1 antibodies to validate protein-level changes predicted from RNA-seq data

• Genomics integration:

  • Correlate DICER1 genotype (e.g., mutations identified in DICER1 syndrome ) with protein expression detected by HRP-conjugated antibodies

  • Analyze how genomic variants affect DICER1 protein levels and localization

• Single-cell applications:

  • Apply HRP-conjugated DICER1 antibodies in single-cell proteomics approaches

  • Correlate with single-cell transcriptomics to understand cell-specific DICER1 regulation

• Spatial omics:

  • Use HRP-conjugated DICER1 antibodies in spatial proteomics approaches

  • Integrate with spatial transcriptomics to map DICER1 expression patterns in tissue context

These integrated approaches provide comprehensive understanding of DICER1 biology beyond what can be achieved with any single methodology.

What emerging techniques might enhance the utility of HRP-conjugated DICER1 antibodies in future research?

Several emerging techniques show promise for expanding DICER1 research capabilities:

• Proximity ligation assays (PLA):

  • Combine HRP-conjugated DICER1 antibodies with antibodies against interaction partners

  • Visualize and quantify specific protein-protein interactions involving DICER1 in situ

• Mass cytometry (CyTOF):

  • Metal-conjugated DICER1 antibodies could enable high-dimensional analysis of DICER1 in conjunction with dozens of other proteins

  • Particularly valuable for heterogeneous samples like tumors or developmental systems

• Expansion microscopy:

  • Physical expansion of samples after labeling with HRP-conjugated DICER1 antibodies

  • Achieve super-resolution imaging with conventional microscopes

• Multiplexed ion beam imaging (MIBI):

  • Metal-conjugated DICER1 antibodies for high-resolution spatial proteomics

  • Map DICER1 expression in relation to tissue architecture with subcellular resolution

• Live-cell temporal studies:

  • While HRP-conjugated antibodies aren't suitable for live cells, complementary approaches using nanobodies or genetically encoded sensors could provide dynamic information

  • Correlate with fixed-time-point antibody studies

• Machine learning analysis:

  • Apply deep learning to images generated with HRP-conjugated DICER1 antibodies

  • Identify subtle patterns in DICER1 expression and localization not apparent to human observers

These emerging techniques could address current limitations in DICER1 research and open new avenues for understanding its complex biology in normal physiology and disease states.

What are the current consensus best practices for working with HRP-conjugated DICER1 antibodies?

Based on the available research and technical information, these consensus best practices emerge:

• Antibody selection:

  • Choose between monoclonal and polyclonal HRP-conjugated DICER1 antibodies based on specific research questions

  • For precise epitope targeting, select monoclonal antibodies with documented epitope specificity (e.g., amino acids 1638-1899 or 1666-1922 )

  • For maximum sensitivity, consider polyclonal antibodies with broad epitope recognition

• Application-specific protocols:

  • Western blotting: Start with 1:1000 dilution , use extended transfer times for DICER1's high molecular weight (~215 kDa)

  • IHC: Use 1:100-500 dilution range , optimize antigen retrieval, and quench endogenous peroxidase

  • IF: Apply at 1:100 dilution with appropriate signal amplification systems

• Validation strategy:

  • Include positive and negative controls for every experiment

  • Validate antibody specificity through multiple approaches (peptide competition, genetic knockdown)

  • Document antibody performance across different experimental systems

• Quantification approaches:

  • Implement standardized quantification methods appropriate for each application

  • For subcellular localization studies, use consistent criteria (e.g., >50% nuclear staining for nuclear localization )

• Documentation and reporting:

  • Maintain detailed records of antibody information, experimental conditions, and validation results

  • Follow established reporting guidelines for antibody-based research in publications

Adhering to these best practices ensures reliable and reproducible results when working with HRP-conjugated DICER1 antibodies across different research applications.

What are the most significant limitations and challenges when working with HRP-conjugated DICER1 antibodies?

Despite their utility, researchers should be aware of these limitations:

• Technical challenges:

  • DICER1's large size (~215 kDa) creates difficulties in protein transfer during Western blotting

  • HRP conjugation may impact antibody binding affinity or specificity compared to unconjugated versions

  • Direct conjugation eliminates amplification steps possible with secondary detection systems

• Biological complexities:

  • DICER1 subcellular localization varies by cell type and physiological state, requiring careful interpretation

  • Post-translational modifications (like phosphorylation at S1712 and S1836 ) may affect antibody recognition

  • Alternative splicing or processing of DICER1 may create variants not detected by all antibodies

• Methodological limitations:

  • HRP-conjugated antibodies are not suitable for applications requiring antibody amplification

  • Not directly applicable to live-cell imaging or flow cytometry without additional modifications

  • Cross-reactivity between species should be validated experimentally despite vendor claims

• Data interpretation challenges:

  • Distinguishing specific from non-specific signals requires rigorous controls

  • Quantitative comparison across different antibodies or detection systems requires careful normalization

  • Correlating protein detection with functional outcomes requires additional experimental approaches