DROSHA Antibody，Biotin conjugated

What is DROSHA and why is it an important target for antibody-based detection?

DROSHA is a Class 2 ribonuclease III enzyme encoded by the DROSHA gene (formerly RNASEN). It functions as a key subunit of the microprocessor protein complex that catalyzes the initial processing step of microRNA (miRNA) synthesis. DROSHA cleaves the stem-loop structure from primary microRNA (pri-miRNA) in the nucleus, yielding precursor miRNA (pre-miRNA), which is then exported to the cytoplasm for further processing .

The importance of DROSHA in miRNA biogenesis makes it a critical target for research into gene regulation pathways. Recent research has also revealed DROSHA's role in DNA damage response and repair mechanisms, particularly in the recruitment of PRC1 component BMI1 at double-strand breaks (DSBs) and subsequent H2A-K119 ubiquitination . This expanded understanding of DROSHA's functions beyond miRNA processing has increased interest in reliable antibody-based detection methods.

What are the fundamental differences between unconjugated and biotin-conjugated DROSHA antibodies?

Unconjugated DROSHA antibodies require secondary detection reagents (such as labeled secondary antibodies) for visualization, while biotin-conjugated DROSHA antibodies have biotin molecules directly attached to the antibody, enabling detection through biotin-binding proteins like streptavidin or avidin .

The key differences include:

The choice between these formats depends on the specific experimental requirements, including sensitivity needs, sample type, and detection method .

What are standard applications for biotin-conjugated DROSHA antibodies?

Biotin-conjugated DROSHA antibodies can be utilized in multiple research applications:

• Western Blotting (WB): Typically used at dilutions of 1:2000-1:16000, providing high sensitivity when coupled with enzyme-conjugated streptavidin detection systems .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Recommended dilutions range from 1:50-1:500, allowing for visualization of DROSHA's nuclear localization .

• Flow Cytometry: Used at dilutions of approximately 1:200-1:1000, enabling quantitative analysis of DROSHA expression in cell populations .

• ELISA: Effective at dilutions of 1:20,000-1:400,000 when using enzyme-conjugated streptavidin, providing exceptional sensitivity for quantitative detection .

• Proximity Ligation Assay (PLA): As demonstrated in DNA damage response studies, biotinylated antibodies allow detection of protein-protein interactions involving DROSHA at DNA repair sites .

For optimal results across these applications, researchers should perform antibody titration experiments to determine the ideal concentration for their specific experimental conditions .

How should I properly biotinylate a DROSHA antibody while maintaining its specificity and affinity?

Proper biotinylation of DROSHA antibodies requires careful consideration of several methodological factors:

Method selection based on research needs:

• Random Conjugation: Uses the ε-amino group of lysine residues or the thiol group of cysteine residues. While simpler, this produces heterogeneous conjugates with variable biotin incorporation .

• Site-Specific Conjugation: More controlled approaches that target specific sites on the antibody:

  • Selenocysteine interface technology introduces unique nucleophilic reactivity at defined positions

  • Enzymatic approaches using transglutaminases or sortases

  • Chemical approaches targeting carbohydrate moieties

Recommended protocol for random biotinylation:

• Dilute purified DROSHA antibody to 4 μM in 100 mM sodium acetate (pH 5.2)

• Add DTT at 0.1 mM followed by biotin-iodoacetamide

• Incubate for 1 hour at room temperature

• Purify using desalting columns or dialysis against PBS

• Validate both biotin incorporation and retained antibody activity

Modern biotinylation kits offer streamlined workflows that can be completed in as little as 10 minutes with high reproducibility, making them suitable for most research applications. These kits typically include all necessary reagents and offer consistent biotin incorporation without requiring purification steps .

What controls should be included when using biotin-conjugated DROSHA antibodies?

Proper experimental design requires several controls to ensure reliable results with biotin-conjugated DROSHA antibodies:

Essential controls:

• Isotype Control: Include an isotype-matched biotinylated control antibody (same species and isotype as your DROSHA antibody) to assess non-specific binding .

• Knockout/Knockdown Validation: Use DROSHA knockout cell lines (such as DROSHA knockout HEK-293T cell line) or DROSHA siRNA knockdown samples to confirm antibody specificity .

• Secondary Reagent Control: Include samples treated only with the streptavidin detection reagent (without primary antibody) to assess background from the detection system .

• Blocking Control: For tissues or cells with endogenous biotin, include samples pre-blocked with avidin/biotin blocking reagents .

• Competitive Binding Control: Pre-incubate the antibody with recombinant DROSHA protein before application to demonstrate specificity .

Example validation data from research:
When validating a biotin-conjugated DROSHA antibody by Western blot, researchers observed a 159 kDa band in wild-type HEK-293T cells that was absent in DROSHA knockout HEK-293T cells, confirming specificity. Similar validation in immunofluorescence showed nuclear localization consistent with DROSHA's known cellular distribution .

How do I troubleshoot high background issues when using biotin-conjugated DROSHA antibodies?

High background is a common challenge when using biotin-conjugated antibodies. Here's a systematic approach to troubleshooting:

Causes and solutions for high background:

• Endogenous Biotin in Samples:

  • Solution: Implement an avidin/biotin blocking step before applying the primary antibody

  • Method: Incubate samples with unconjugated avidin followed by excess biotin to block endogenous biotin

• Excessive Antibody Concentration:

  • Solution: Perform titration experiments to determine optimal antibody concentration

  • Method: Test serial dilutions (e.g., 1:100, 1:500, 1:1000, 1:5000) to identify the concentration that gives specific signal with minimal background

• Non-specific Binding:

  • Solution: Optimize blocking and wash conditions

  • Method: Increase blocking time/concentration (typically 3-5% BSA or 5-10% normal serum), add 0.1-0.3% Triton X-100 to washes, and increase wash duration/number

• Over-biotinylation of Antibody:

  • Solution: Use antibodies with optimal biotin:antibody ratio or reduce biotinylation time

  • Method: Commercial antibodies typically have optimized ratios; for custom biotinylation, reduce reaction time or reagent concentration

• Cross-reactivity:

  • Solution: Pre-adsorb the antibody or use more specific antibody clones

  • Method: Pre-incubate antibody with lysates from knockout cells or recombinant proteins of related family members

How can biotin-conjugated DROSHA antibodies be used to study DROSHA's role in DNA damage response?

Recent research has revealed DROSHA's critical involvement in DNA damage response through mechanisms distinct from its role in miRNA processing. Biotin-conjugated DROSHA antibodies offer powerful tools for investigating these processes:

Methodological approach:

• Proximity Ligation Assay (PLA):

  • Combine anti-biotin antibody with anti-BMI1 antibody to detect DROSHA-BMI1 interactions at DNA damage sites

  • This approach revealed that DROSHA and DICER control the recruitment of BMI1 at double-strand breaks (DSBs)

• Chromatin Immunoprecipitation (ChIP):

  • Use biotin-conjugated DROSHA antibodies to immunoprecipitate DROSHA-bound chromatin

  • Couple with sequencing (ChIP-seq) to map DROSHA binding sites at DNA damage loci

• RNA-Immunoprecipitation (RIP):

  • Apply biotin-conjugated DROSHA antibodies to immunoprecipitate DROSHA-bound RNAs

  • This approach showed that BMI1 associates with damage-induced long non-coding RNAs (dilncRNAs) in a DROSHA-dependent manner

Key research findings:
Studies using these approaches demonstrated that DROSHA and DICER control transcriptional silencing of genes adjacent to damaged chromatin through a mechanism involving BMI1 recruitment and H2A-K119 ubiquitination . This function appears to be independent of DROSHA's canonical miRNA processing role and provides new insights into DNA damage-induced transcriptional regulation.

What are the advantages and limitations of using F(ab')2 fragments versus whole IgG for biotin-conjugated DROSHA antibodies?

The choice between F(ab')2 fragments and whole IgG for biotin-conjugated DROSHA antibodies significantly impacts experimental outcomes:

Comparative analysis:

Application-specific considerations:

• Live Cell Applications: F(ab')2 fragments are preferred because they avoid binding to Fc receptors on live cells, reducing non-specific signal .

• Tissue Immunohistochemistry: F(ab')2 fragments offer better penetration in dense tissues but may provide lower sensitivity than whole IgG .

• Proximity-based Assays: F(ab')2 fragments place detection tags closer to the target epitope, potentially improving spatial resolution in techniques like PLA .

• Storage Stability: Whole IgG typically exhibits superior shelf-life, with biotin-conjugated F(ab')2 fragments requiring more careful storage conditions (recommended: aliquot and freeze at -70°C or add equal volume of glycerol for storage at -20°C) .

How does MDM2-mediated ubiquitination of DROSHA affect epitope accessibility for antibody detection?

MDM2 has been identified as an E3 ligase for DROSHA, mediating its ubiquitination and affecting its function in response to cellular environmental changes. This post-translational modification can impact antibody-based detection:

Mechanistic impact on epitope accessibility:

• Ubiquitination Sites and Epitope Masking:

  • MDM2-mediated ubiquitination of DROSHA can potentially mask epitopes, particularly in regions where ubiquitin chains are attached

  • In vitro ubiquitination assays have demonstrated that MDM2 has E3 ligase activity for DROSHA, resulting in specific ubiquitin attachment patterns

• Conformational Changes:

  • Ubiquitination may induce conformational changes in DROSHA protein structure

  • These changes can expose or conceal epitopes, affecting antibody binding efficiency

Experimental strategies to address this challenge:

• Epitope Mapping:

  • Map antibody epitopes relative to known ubiquitination sites on DROSHA

  • Select antibodies targeting regions less likely to be affected by ubiquitination

• Deubiquitination Treatment:

  • Include deubiquitinating enzyme treatment conditions in parallel experiments

  • Compare antibody detection between ubiquitinated and deubiquitinated samples

• Multiple Antibody Approach:

  • Use multiple antibodies targeting different DROSHA epitopes

  • This provides complementary data to overcome potential epitope masking issues

Research has shown that silencing MDM2 greatly reduces ubiquitinated DROSHA levels, suggesting that ubiquitination status monitoring may be important when studying DROSHA in different cellular contexts .

How do biotin-conjugated DROSHA antibodies compare to other detection methods in sensitivity and specificity?

When selecting detection methods for DROSHA research, it's important to understand the comparative advantages of different approaches:

Sensitivity comparison across detection methods:

Method-specific considerations:

• Biotin-Streptavidin Systems:

  • Advantage: Signal amplification through multiple biotin-streptavidin interactions increases sensitivity

  • Limitation: Potential endogenous biotin interference in certain sample types

  • Optimal for: Western blots (1:2000-1:16000 dilution) and ELISA (1:20,000-1:400,000 dilution)

• Direct Fluorophore Conjugation:

  • Advantage: Simplified workflow and excellent for multiplexing

  • Limitation: Limited signal amplification compared to biotin-streptavidin

  • Optimal for: Immunofluorescence microscopy and flow cytometry

• Enzyme-based Detection:

  • Advantage: Extreme sensitivity through enzymatic amplification

  • Limitation: Limited dynamic range and poor spatial resolution

  • Optimal for: Western blots and immunohistochemistry

Research data indicates that biotin-conjugated antibodies with streptavidin detection systems can offer up to 4-8 fold signal enhancement compared to direct conjugation methods, particularly when using enzymes like alkaline phosphatase with streptavidin .

What are the considerations for choosing between polyclonal and monoclonal biotin-conjugated DROSHA antibodies?

The choice between polyclonal and monoclonal biotin-conjugated DROSHA antibodies significantly impacts experimental outcomes:

Comparative analysis:

Application-specific recommendations:

• For Western Blotting:

  • Polyclonal antibodies often provide stronger signals (recommended dilutions: 1:2000-1:16000)

  • Example: Proteintech's polyclonal anti-DROSHA (27958-1-AP) shows excellent specificity at 150 kDa in multiple human cell lines

• For Immunoprecipitation:

  • Monoclonal antibodies typically offer cleaner results with less background

  • Example: Abcam's EPR12794 monoclonal antibody (ab183732) demonstrates strong specificity in IP applications

• For Immunofluorescence:

  • Both types can work well; selection depends on specific epitope accessibility

  • U2OS cells have been successfully used to validate DROSHA antibodies in IF applications

• For Flow Cytometry:

  • Monoclonal antibodies often provide more consistent results

  • Example flow data: Boster's A00111-3 antibody has been validated in HL-60 cells

When possible, validation in DROSHA knockout systems (such as DROSHA knockout HEK-293T cell lines) provides the strongest evidence for antibody specificity regardless of antibody type .

How can I integrate biotin-conjugated DROSHA antibodies into multi-parameter experimental designs?

Biotin-conjugated DROSHA antibodies offer versatility for integration into complex experimental designs studying multiple parameters simultaneously:

Strategic approaches for multi-parameter studies:

• Sequential Detection Protocols:

  • Apply biotin-conjugated DROSHA antibody first, detect with streptavidin-conjugate

  • Block remaining biotin/streptavidin binding sites

  • Apply subsequent directly-conjugated antibodies for other targets

  • Example application: Studying DROSHA and DICER co-localization at DNA damage sites

• Proximity Ligation Assay (PLA) Integration:

  • Use biotin-conjugated DROSHA antibody with antibodies against potential interaction partners

  • This approach revealed DROSHA-BMI1 interactions in DNA damage contexts

  • Protocol outline: Combine anti-biotin antibody with anti-BMI1 antibody to visualize proximity

• Multiplexed Flow Cytometry:

  • Use streptavidin conjugated to compatible fluorophores (e.g., PE)

  • Combine with directly conjugated antibodies for other targets

  • Example: DROSHA detection alongside cell cycle markers or other RNA processing proteins

• ChIP-seq Analysis:

  • Use biotin-conjugated DROSHA antibodies for chromatin immunoprecipitation

  • Integrate with other epigenetic marks (H2A-K119ub, γH2AX) to build comprehensive maps of DROSHA activity at chromatin

  • This approach helped establish DROSHA's role in transcriptional silencing at DNA damage sites

These integrated approaches have revealed that DROSHA works alongside DICER to control BMI1 recruitment to DNA damage sites and subsequent chromatin modifications, providing new insights into non-canonical DROSHA functions beyond miRNA processing .

How might advances in site-specific biotinylation improve DROSHA antibody applications?

Emerging technologies in site-specific biotinylation promise to enhance DROSHA antibody performance and expand application possibilities:

Innovative approaches and their potential impact:

• Selenocysteine Interface Technology:

  • Introduces the 21st natural amino acid selenocysteine into antibodies

  • Creates unique nucleophilic reactivity for site-specific conjugation

  • Advantages over conventional methods:

    • Minor modification at the C-terminus that doesn't interfere with disulfide bridges

    • No activation requirement

    • Precise 1:1 stoichiometry of biological and chemical components

• Enzymatic Biotinylation:

  • Utilizes enzymes like bacterial biotin ligase (BirA) for site-specific attachment

  • Requires genetic incorporation of recognition sequences

  • Offers exceptional control over biotinylation sites and stoichiometry

• Click Chemistry Approaches:

  • Incorporates non-natural amino acids with azide or alkyne groups

  • Enables bioorthogonal conjugation with biotin derivatives

  • Provides clean, highly specific biotinylation under mild conditions

These advances could significantly improve DROSHA antibody applications by:

• Enhancing sensitivity through optimal biotin positioning

• Improving batch-to-batch consistency for more reproducible results

• Preserving full antibody functionality by avoiding modification of critical regions

• Enabling precise control of biotin:antibody ratio for optimal signal:noise

What emerging applications might benefit from biotin-conjugated DROSHA antibodies?

Several cutting-edge research areas could significantly benefit from advanced biotin-conjugated DROSHA antibodies:

Promising research directions:

• Single-Cell Proteomics:

  • Application: Using biotin-conjugated DROSHA antibodies with mass cytometry (CyTOF)

  • Benefit: Enables comprehensive profiling of DROSHA expression and associated proteins at single-cell resolution

  • Potential insight: Identification of cell subpopulations with unique DROSHA activity patterns

• Spatial Transcriptomics Integration:

  • Application: Combining biotin-conjugated DROSHA antibodies with in situ RNA detection

  • Benefit: Maps spatial relationships between DROSHA protein localization and miRNA/mRNA expression

  • Potential insight: Understanding tissue-specific regulation of RNA processing

• Super-Resolution Microscopy:

  • Application: Using biotin-conjugated DROSHA antibodies with quantum dot-labeled streptavidin

  • Benefit: Achieves nanometer-scale resolution of DROSHA localization at specific nuclear substructures

  • Potential insight: Detailed understanding of DROSHA's dynamic localization during DNA damage response

• CRISPR Screens Combined with DROSHA Detection:

  • Application: Using biotin-conjugated DROSHA antibodies to assess DROSHA function/localization in CRISPR-edited cells

  • Benefit: High-throughput assessment of genetic factors influencing DROSHA activity

  • Potential insight: Identification of novel regulators of DROSHA function in both canonical and non-canonical pathways

• Liquid Biopsy Development:

  • Application: Using biotin-conjugated DROSHA antibodies to detect DROSHA in extracellular vesicles

  • Benefit: Potential biomarker development for cancer and other diseases

  • Potential insight: Understanding how altered DROSHA function contributes to disease pathogenesis