DROSHA Antibody,HRP conjugated

What is DROSHA and why is it important in research?

DROSHA (Ribonuclease III) is a crucial enzyme in microRNA (miRNA) biogenesis, functioning as part of the microprocessor complex along with its partner DGCR8. It plays a critical role in the initial processing of primary miRNAs in the nucleus. Research interest in DROSHA stems from its essential function in gene regulation through the miRNA pathway, with implications in development, cellular differentiation, and various disease states. DROSHA has a molecular weight of approximately 159 kDa and is encoded by the RNASEN gene (also known by alias symbols RN3, ETOHI2, RNASEN, RANSE3L, RNASE3L, and HSA242976) .

What applications are HRP-conjugated DROSHA antibodies suitable for?

HRP-conjugated DROSHA antibodies are primarily designed for Western blotting (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications. For Western blot applications, these antibodies are typically used at dilutions of 1:500-1:1000, while ELISA applications typically employ higher dilutions of approximately 1:10000 . The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows and potentially reducing background signal. These antibodies have been validated for detecting endogenous levels of DROSHA protein in human and mouse samples .

What are the optimal conditions for Western blot detection of DROSHA using HRP-conjugated antibodies?

For optimal Western blot detection of DROSHA using HRP-conjugated antibodies, several technical parameters should be considered. Sample preparation should include approximately 50 μg of protein per lane, with separation performed on 5-20% SDS-PAGE gels at 70-90V for 2-3 hours. After electrophoresis, proteins should be transferred to a nitrocellulose or PVDF membrane at 150mA for 50-90 minutes. Blocking should be performed using 5% non-fat milk in TBS for 1.5 hours at room temperature. The HRP-conjugated DROSHA antibody should be applied at the recommended dilution (typically 1:500-1:1000 for Western blots) and incubated overnight at 4°C. Following washing with TBS-0.1% Tween (three times for 5 minutes each), detection can be performed using an enhanced chemiluminescent (ECL) detection system. DROSHA is expected to appear as a specific band at approximately 159-200 kDa, with some variation in apparent molecular weight possibly due to post-translational modifications or detection of different isoforms .

How can I design experiments to detect different DROSHA isoforms?

Designing experiments to detect different DROSHA isoforms requires careful consideration of antibody selection and experimental controls. Research has identified alternatively spliced DROSHA transcripts with differential subcellular localization patterns. For instance, the Dr1 isoform localizes to both nuclear and cytoplasmic compartments, while the Dr2 isoform shows exclusively nuclear localization .

To distinguish between isoforms:

• Select cell lines with known differential expression patterns of DROSHA isoforms. For example, U2OS, NT2, and HEK293 cells show higher Dr2/Dr1 ratios (nuclear-only pattern), while NCI-H1703 and PC9 cells show lower ratios (nuclear and cytoplasmic pattern) .

• Perform RT-PCR or RT-qPCR to validate the expression patterns of different isoforms at the mRNA level before protein analysis.

• Combine immunofluorescence microscopy with cellular fractionation and Western blotting to confirm the subcellular distribution patterns of DROSHA isoforms.

• When interpreting results, consider that the apparent molecular weight of DROSHA may vary from the predicted 159 kDa, with some antibodies detecting bands at approximately 200 kDa .

What controls should be included when using DROSHA antibodies in experimental workflows?

Robust experimental design with appropriate controls is essential when using DROSHA antibodies:

Positive Controls: Include cell lines with known DROSHA expression, such as:

• Human cell lines: HeLa, HEK293, Jurkat, HepG2, SW620, K562

• Mouse cell lines: Neuro-2a, Ana-1

Negative Controls:

• Antibody specificity can be validated through knockdown experiments using siRNA or shRNA targeting DROSHA .

• Include isotype control antibodies (e.g., rabbit IgG at equivalent concentration) to assess non-specific binding.

• For flow cytometry applications, include unlabeled samples without primary and secondary antibody incubation as blank controls .

Loading Controls:

• For Western blotting, include detection of housekeeping proteins such as Actin, HSP70, or other stable reference proteins to normalize DROSHA expression levels .

Fractionation Controls:

• When analyzing subcellular localization, include markers for specific cellular compartments to validate fractionation efficiency .

How can I verify the specificity of DROSHA antibody detection in my experimental system?

Verifying antibody specificity is crucial for generating reliable data. For DROSHA antibodies, several validation approaches can be employed:

• Knockdown Validation: Perform siRNA or shRNA-mediated knockdown of DROSHA and confirm reduced signal intensity in Western blot or immunofluorescence applications. This approach has been successfully used to demonstrate the specificity of DROSHA antibodies in previous studies .

• Multiple Antibody Validation: Use multiple antibodies targeting different epitopes of DROSHA to confirm consistent detection patterns. For instance, comparing results from antibodies that recognize the N-terminal versus C-terminal regions of DROSHA can help validate specificity.

• Cell Line Panel Analysis: Test the antibody across multiple cell lines with varying DROSHA expression levels. Consistent detection patterns that correlate with known expression levels provide evidence for specificity. Cell lines such as HeLa, HEK293, Jurkat, HepG2, SW620, K562 (human) and Neuro-2a, Ana-1 (mouse) have been validated for DROSHA expression analysis .

• Isoform Expression Analysis: Compare antibody detection patterns with mRNA expression data for different DROSHA isoforms. Correlation between protein detection patterns and transcript levels provides additional evidence for specificity .

• Band Size Verification: Confirm that the detected band appears at the expected molecular weight (approximately 159-200 kDa for DROSHA), with consideration for potential post-translational modifications that may affect migration patterns .

What are common technical issues when using HRP-conjugated antibodies and how can they be addressed?

HRP-conjugated antibodies present specific technical challenges that researchers should anticipate and address:

• High Background Signal:

  • Cause: Insufficient blocking, excessive antibody concentration, or non-specific binding

  • Solution: Optimize blocking conditions (try different blocking agents like BSA or casein if milk protein is ineffective), increase washing duration/frequency, and titrate antibody concentration to determine optimal dilution

• Loss of Signal Over Time:

  • Cause: HRP degradation due to improper storage or repeated freeze-thaw cycles

  • Solution: Store antibodies in light-protected vials, aliquot stock solutions to minimize freeze-thaw cycles, and consider adding 50% glycerol for long-term storage at -20°C to -80°C

• Variable Signal Intensity:

  • Cause: Inconsistent transfer efficiency or protein loading

  • Solution: Verify transfer efficiency with reversible protein stains, normalize to loading controls, and ensure consistent sample preparation protocols

• False Positive Signals:

  • Cause: Cross-reactivity with similar proteins or endogenous peroxidase activity

  • Solution: Include appropriate negative controls, consider quenching endogenous peroxidase activity with hydrogen peroxide treatment before antibody incubation, and validate with knockdown experiments

• Weak or Absent Signal:

  • Cause: Insufficient protein quantity, protein degradation, or inefficient transfer

  • Solution: Increase protein loading (50 μg recommended for DROSHA detection), add protease inhibitors during sample preparation, and optimize transfer conditions for high molecular weight proteins like DROSHA

How does caspase-mediated cleavage of DROSHA affect antibody detection patterns?

Research has demonstrated that DROSHA is cleaved by caspases during apoptosis, which can significantly impact antibody detection patterns . This cleavage generates distinct fragments that may be detected differently depending on the epitope recognized by the antibody.

When conducting experiments involving apoptotic conditions or stress responses:

• Multiple Fragment Detection: DROSHA antibodies may detect both the full-length protein (159-200 kDa) and smaller cleavage fragments in apoptotic samples.

• Time-Course Considerations: In apoptosis-inducing treatments, the intensity of the full-length DROSHA band may diminish over time with a corresponding increase in cleavage products.

• Antibody Selection Strategy: Use antibodies targeting epitopes outside known caspase cleavage sites if detection of intact DROSHA is the primary goal. Conversely, antibodies recognizing regions that include caspase cleavage sites can be useful for monitoring DROSHA processing during apoptosis.

• Control Inclusion: Include positive controls for apoptosis induction such as cleaved caspase-3 detection when investigating potential DROSHA cleavage events.

• Result Interpretation: When unexpected band patterns are observed, consider whether experimental conditions might have induced apoptosis, as this could explain the presence of DROSHA cleavage products .

How can DROSHA antibodies be used to investigate microRNA processing dysfunction in disease models?

DROSHA antibodies provide valuable tools for investigating miRNA processing dysfunction in various disease models, particularly in cancer, neurological disorders, and developmental syndromes where miRNA dysregulation is implicated:

• Quantitative Analysis of DROSHA Expression:

  • Western blotting with HRP-conjugated DROSHA antibodies enables quantitative comparison of DROSHA protein levels across different disease models and control tissues

  • Differential expression patterns may correlate with altered miRNA profiles and disease phenotypes

• Subcellular Localization Studies:

  • Immunofluorescence microscopy using DROSHA antibodies can reveal altered localization patterns in disease states

  • Changes in nuclear-cytoplasmic distribution of DROSHA may indicate dysfunction in miRNA processing pathways, as different isoforms show distinct localization patterns

• Protein-Protein Interaction Analysis:

  • Co-immunoprecipitation experiments using DROSHA antibodies can identify alterations in the composition of the microprocessor complex (DROSHA-DGCR8) in disease models

  • Changes in interactions with regulatory proteins may explain processing defects in specific diseases

• Analysis of Post-translational Modifications:

  • Western blotting with DROSHA antibodies combined with treatments affecting specific modifications can reveal regulatory mechanisms impaired in disease states

  • Phosphorylation, acetylation, or ubiquitination may affect DROSHA function and stability

• Investigation of Cleavage Events:

  • Detection of DROSHA cleavage products using specific antibodies can provide insights into regulatory mechanisms involving proteolytic processing in disease progression

  • Caspase-mediated cleavage of DROSHA during apoptosis may contribute to miRNA processing dysfunction in diseases involving cellular stress responses

How does alternative splicing affect DROSHA localization and how can this be studied?

Alternative splicing of DROSHA transcripts generates isoforms with distinct subcellular localization patterns that impact miRNA processing efficiency. Research has identified that alternatively spliced DROSHA transcripts lacking portions of the arginine/serine-rich (RS-rich) domain show differential localization patterns .

To investigate this phenomenon:

What technical approaches can be used to differentiate between active and inactive forms of DROSHA?

Distinguishing between active and inactive forms of DROSHA is crucial for understanding regulatory mechanisms affecting miRNA processing. Several technical approaches can be employed:

• In Vitro Processing Assays:

  • Immunoprecipitate DROSHA using specific antibodies followed by incubation with radiolabeled primary miRNA substrates

  • Measure processing efficiency through gel electrophoresis and autoradiography to detect cleavage products

  • Compare processing activity across different experimental conditions or sample types

• Phosphorylation State Analysis:

  • Use phospho-specific antibodies in conjunction with general DROSHA antibodies to detect phosphorylated forms that may correlate with altered activity

  • Combine with phosphatase treatments to confirm the role of phosphorylation in regulating DROSHA activity

  • Correlate phosphorylation patterns with processing efficiency of target miRNAs

• Complex Formation Assessment:

  • Analyze DROSHA interaction with essential cofactors like DGCR8 using co-immunoprecipitation followed by Western blotting

  • Size exclusion chromatography combined with Western blotting can separate different DROSHA-containing complexes

  • Native gel electrophoresis followed by antibody detection can preserve and detect intact complexes with different compositions

• Subcellular Fractionation:

  • Separate nuclear and cytoplasmic fractions to correlate DROSHA localization with activity

  • Different isoforms show distinct localization patterns that may reflect functional differences

  • Combine with processing assays to determine which cellular compartment contains active DROSHA

• Structural Analysis Approach:

  • Use limited proteolysis followed by antibody detection to assess conformational states that may correspond to active versus inactive forms

  • Conformational changes affecting epitope accessibility can be detected using panels of antibodies targeting different regions of DROSHA

What methodological approaches can differentiate between DROSHA isoforms with different subcellular localizations?

Differentiating between DROSHA isoforms with distinct subcellular localizations requires a multi-faceted approach:

• RT-PCR and RT-qPCR Analysis:

  • Design primers spanning alternative splice junctions to specifically amplify different DROSHA isoform transcripts

  • Calculate the ratio of nuclear (Dr2) to nuclear-cytoplasmic (Dr1) isoforms to predict predominant localization patterns

  • This initial analysis guides subsequent protein-level investigations

• Cellular Fractionation Combined with Western Blotting:

  • Separate nuclear and cytoplasmic fractions using optimized protocols

  • Use Western blotting with DROSHA antibodies to detect isoform distribution across fractions

  • Include fraction-specific markers (e.g., nuclear lamins, cytoplasmic tubulin) to validate fractionation quality

  • Quantify the relative abundance of DROSHA in different cellular compartments

• Immunofluorescence Microscopy:

  • Fix cells using paraformaldehyde and perform immunostaining with DROSHA antibodies

  • Use confocal laser scanning microscopy to visualize subcellular distribution patterns

  • Perform co-localization analysis with nuclear and cytoplasmic markers

  • This approach provides spatial resolution that complements biochemical fractionation

• Cell Line Selection Strategy:

  • Use cell lines with known differential expression patterns of DROSHA isoforms

  • The cellular distribution pattern correlates with the ratio of isoform expression:

  Cell Line	DROSHA Distribution	Dr2/Dr1 Ratio	Detection Method
  U2OS	Nuclear only	High	IF microscopy
  NT2	Nuclear only	High	IF microscopy
  HEK293	Nuclear only	High	IF + Western
  NCI-H1703	Nuclear + Cytoplasmic	Low	IF + Western
  PC9	Nuclear + Cytoplasmic	Low	IF + Western

• Validation Through Knockdown and Rescue:

  • Perform siRNA knockdown of endogenous DROSHA followed by expression of specific isoforms

  • This approach confirms the localization patterns observed for individual isoforms

  • Include antibody specificity validation through knockdown experiments

How can researchers interpret unexpected band patterns in Western blots using DROSHA antibodies?

When encountering unexpected band patterns in Western blots using DROSHA antibodies, systematic analysis can help determine their significance:

• Multiple Band Patterns:

  • Expected molecular weight of full-length DROSHA is approximately 159 kDa, but it may appear at 200 kDa in some gel systems

  • Additional bands may represent:
    a. Alternative splice variants (e.g., Dr1, Dr2, Dr3)
    b. Post-translationally modified forms (phosphorylation, ubiquitination)
    c. Proteolytic cleavage products, particularly in apoptotic samples
    d. Cross-reactivity with related proteins

• Analytical Approach:

  • Compare observed patterns with predicted molecular weights of known DROSHA isoforms

  • Consider experimental conditions that might induce proteolytic processing (e.g., apoptosis triggers)

  • Verify with positive control samples (e.g., HeLa, HEK293, Jurkat cell lysates)

  • Confirm specificity through knockdown experiments targeting DROSHA

• Technical Considerations:

  • Sample preparation methods can affect band patterns (e.g., protease inhibitor inclusion)

  • Gel percentage and running conditions influence resolution of high molecular weight proteins

  • Transfer efficiency can vary for different molecular weight ranges

  • Blocking conditions and antibody dilutions may affect detection sensitivity of minor isoforms

• Validation Strategies:

  • Use multiple antibodies targeting different epitopes of DROSHA to confirm consistent patterns

  • Combine with RT-PCR analysis to correlate protein bands with transcript variants

  • Consider mass spectrometry analysis for unambiguous identification of unexpected bands

• Interpretation Guidelines:

  Band Size (kDa)	Potential Identity	Validation Approach
  159-200	Full-length DROSHA	Knockdown, positive controls
  110-130	Possible cleavage product	Apoptosis induction, caspase inhibitors
  65-85	Possible cleavage product	Apoptosis induction, caspase inhibitors
  Variable	Splice variants	RT-PCR correlation

What considerations are important when designing experiments to investigate DROSHA's role in apoptotic pathways?

When investigating DROSHA's role in apoptotic pathways, several key experimental design considerations are essential:

• Time-Course Analysis:

  • DROSHA undergoes caspase-mediated cleavage during apoptosis, necessitating temporal analysis

  • Design experiments with multiple time points following apoptosis induction

  • Early time points capture initial cleavage events, while later points show complete processing

  • Western blotting with DROSHA antibodies at different time points can track the progression of cleavage

• Apoptosis Induction Methods:

  • Select appropriate apoptosis triggers based on research context (e.g., staurosporine, TNF-α, Fas ligand)

  • Include both intrinsic and extrinsic pathway activators to comprehensively assess DROSHA processing

  • Titrate apoptosis inducers to achieve controlled, synchronous apoptosis progression

• Caspase Inhibition Studies:

  • Include specific caspase inhibitors (e.g., Z-VAD-FMK) to confirm the role of caspases in DROSHA cleavage

  • Compare DROSHA processing patterns in the presence and absence of inhibitors

  • This approach can identify specific caspases responsible for DROSHA cleavage

• Concurrent Detection of Multiple Proteins:

  • Simultaneously analyze DROSHA with other miRNA processing components (Dicer, DGCR8, TRBP2)

  • All these components undergo caspase-mediated cleavage during apoptosis

  • Include detection of established apoptosis markers (cleaved caspase-3, PARP cleavage)

  • This comprehensive approach provides context for DROSHA processing events

• Functional Analysis:

  • Correlate DROSHA cleavage with changes in miRNA processing efficiency

  • Measure levels of primary, precursor, and mature miRNAs at different stages of apoptosis

  • This connects proteolytic events to functional outcomes in the miRNA pathway

• Technical Recommendations:

  Analysis Approach	Purpose	Specific Techniques
  Protein detection	Track DROSHA cleavage	Western blot with antibodies detecting different epitopes
  Activity assessment	Measure functional impact	In vitro processing assays with immunoprecipitated DROSHA
  Localization changes	Assess redistribution	Immunofluorescence microscopy, subcellular fractionation
  miRNA profiling	Evaluate downstream effects	qPCR, small RNA sequencing
  Mechanistic validation	Confirm causality	Site-directed mutagenesis of caspase cleavage sites