ICE2 Antibody, Biotin conjugated

What is ICE2 protein and why is it significant in research?

ICE2 (also known as BRCC1, NARG2, UNQ3101/PRO10100) functions as a component of the Little Elongation Complex (LEC). This complex plays a critical role in regulating small nuclear RNA (snRNA) gene transcription by RNA polymerase II and III . The significance of ICE2 in research stems from its involvement in fundamental transcriptional regulatory mechanisms. Understanding ICE2 function can provide insights into RNA processing disorders and potential therapeutic targets for related diseases. Methodologically, researchers often use antibodies against ICE2 to study its expression patterns, protein interactions, and subcellular localization.

What are the common aliases and identifiers for ICE2 protein?

When conducting literature searches or database queries, researchers should be aware of multiple identifiers for the ICE2 protein:

Using these alternative identifiers will ensure comprehensive literature searches and avoid missing relevant publications when researching ICE2 .

What is the advantage of using a biotin-conjugated antibody for ICE2 detection?

Biotin-conjugated antibodies offer several methodological advantages for ICE2 detection:

• Signal amplification: The biotin-avidin system provides significant signal enhancement due to the high affinity between biotin and streptavidin/avidin (Kd ≈ 10^-15 M), improving detection sensitivity .

• Versatility in detection systems: Biotin conjugates can be detected using various avidin/streptavidin-coupled reporter molecules (HRP, fluorophores, gold particles) .

• Compatibility with multiple techniques: A single biotin-conjugated primary antibody can be used across different applications including ELISA, immunohistochemistry, and western blotting .

• Reduced background in multi-labeling experiments: Biotin-conjugated antibodies allow for sequential detection protocols, reducing cross-reactivity issues common in simultaneous multi-antibody incubations.

What applications is the ICE2 antibody, biotin conjugated suitable for?

• Western Blotting: While not explicitly validated, biotin-conjugated antibodies generally perform well in western blotting when used with streptavidin-HRP detection systems .

• Immunohistochemistry/Immunocytochemistry: Test with appropriate biotin-blocking steps and streptavidin-based detection systems. Optimization of antigen retrieval methods may be necessary .

• Flow Cytometry: Consider using with streptavidin-fluorophore conjugates, but validate specificity and signal-to-noise ratio .

• Immunoprecipitation: May require testing with magnetic streptavidin beads for pull-down experiments.

When adapting this antibody to non-validated applications, researchers should perform appropriate controls including no-primary antibody, isotype controls, and where possible, positive and negative tissue/cell controls.

How can epitope mapping be performed to characterize the binding site of ICE2 antibody?

Epitope mapping for ICE2 antibody can be approached through several methodological strategies:

• Peptide Array Analysis: Synthesize overlapping peptides spanning the ICE2 protein (focusing on the 876-982AA region used as immunogen) and test antibody binding to identify the minimal epitope sequence .

• Alanine Scanning Mutagenesis: Create point mutations in recombinant ICE2 protein where each residue is systematically replaced with alanine, then test antibody binding to identify critical binding residues .

• Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): This approach can identify protected regions of the protein upon antibody binding, providing structural information about the epitope .

• X-ray Crystallography or Cryo-EM: For definitive epitope characterization, co-crystallize the antibody Fab fragment with ICE2 protein or use Cryo-EM to visualize the complex, similar to the approach used for SARS-CoV-2 antibodies .

Data interpretation should account for conformational versus linear epitopes, as the recognition pattern will influence antibody performance across different applications.

What strategies can resolve cross-reactivity issues with ICE2 antibody in multi-protein complex analyses?

When studying ICE2 within the Little Elongation Complex (LEC), researchers may encounter cross-reactivity challenges. Several methodologies can address these issues:

• Antibody Validation with Knockout/Knockdown Controls: Generate ICE2 knockout or knockdown cells/tissues to confirm antibody specificity and identify any cross-reactive bands or signals .

• Sequential Immunoprecipitation:

  • First round: Precipitate with ICE2 antibody

  • Elution under mild conditions

  • Second round: Re-precipitate with antibodies against suspected cross-reactive proteins

  • Analysis of fractions can distinguish between true interactions and cross-reactivity

• Competitive Binding Assays: Pre-incubate the antibody with recombinant ICE2 protein before application to samples. Specific signals should be blocked while cross-reactive signals may persist .

• Epitope-blocking Peptides: Use synthetic peptides corresponding to the ICE2 epitope to confirm signal specificity.

• Orthogonal Detection Methods: Combine biotin-conjugated antibody detection with mass spectrometry validation to confirm protein identity beyond antibody recognition.

How can researchers optimize ICE2 antibody-based chromatin immunoprecipitation (ChIP) protocols?

Optimizing ChIP protocols for ICE2 antibody requires addressing several technical considerations:

• Crosslinking Optimization:

  • Test both formaldehyde (1-3%) and dual crosslinking (DSG followed by formaldehyde) to capture indirect DNA interactions via protein complexes

  • Optimize crosslinking time (5-20 minutes) to balance efficiency and reversibility

• Sonication Parameters:

  • Aim for chromatin fragments of 200-500bp for high resolution

  • Optimize sonication cycles (typically 10-30 cycles of 30s on/30s off) for consistent fragmentation

  • Verify fragment size by agarose gel electrophoresis

• Antibody Incubation Conditions:

  • Determine optimal antibody concentration through titration experiments (typically 2-10μg per ChIP reaction)

  • Test both overnight incubation at 4°C and shorter incubations (4-6 hours)

  • Include appropriate IgG control and positive control antibody (e.g., against histone marks)

• Biotin-Streptavidin Interaction Utilization:

  • Leverage the biotin conjugation for streptavidin bead capture

  • Consider pre-clearing samples with streptavidin beads to reduce background

  • Test elution conditions that preserve biotin-streptavidin interaction while releasing DNA-protein complexes

• Sequential ChIP:

  • For studying co-occupancy with other LEC components, develop sequential ChIP protocols

  • First ChIP with ICE2 antibody, followed by elution and second ChIP with antibodies against other complex components

What are the considerations for using the biotin-conjugated ICE2 antibody in proximity ligation assays (PLA)?

Proximity ligation assays can provide valuable insights into ICE2 protein interactions within the cellular context. When adapting biotin-conjugated ICE2 antibody for PLA, researchers should consider:

• Antibody Compatibility Strategy:

  • Select a compatible secondary antibody system that works with biotin-conjugated primary antibodies

  • Consider using streptavidin-conjugated PLA probes to directly detect the biotin-conjugated ICE2 antibody

  • For dual-recognition PLA, pair with non-biotinylated antibodies against potential interaction partners

• Endogenous Biotin Interference Management:

  • Include biotin blocking steps, particularly for biotin-rich tissues (liver, kidney)

  • Pre-treat samples with streptavidin followed by biotin to block endogenous biotin

  • Include appropriate negative controls to assess background from endogenous biotin

• Signal-to-Noise Optimization:

  • Titrate antibody concentration to minimize background while maintaining specific signal

  • Optimize fixation conditions to preserve epitope accessibility

  • Include competition controls with recombinant ICE2 protein to confirm signal specificity

• Multi-color PLA Development:

  • Design strategies for simultaneously visualizing multiple protein interactions

  • Combine PLA with conventional immunofluorescence for contextual information

How should researchers validate the specificity of ICE2 antibody for their particular experimental system?

Rigorous validation is essential before using ICE2 antibody in critical experiments. A comprehensive validation approach includes:

• Genetic Validation:

  • Use CRISPR/Cas9 knockout or siRNA knockdown of ICE2

  • Compare antibody signal between wild-type and ICE2-depleted samples

  • Expected outcome: Significant reduction or disappearance of specific signal

• Overexpression Validation:

  • Transfect cells with tagged ICE2 expression construct

  • Perform dual labeling with anti-tag antibody and ICE2 antibody

  • Expected outcome: Co-localization of signals and increased intensity in transfected cells

• Peptide Competition:

  • Pre-incubate antibody with excess immunizing peptide (876-982AA region)

  • Apply to parallel samples alongside non-blocked antibody

  • Expected outcome: Specific signal should be abrogated by peptide competition

• Western Blot Analysis:

  • Verify presence of a predominant band at the expected molecular weight (~111 kDa)

  • Check for absence of non-specific bands

  • Compare band pattern across multiple cell lines with known ICE2 expression levels

• Cross-platform Validation:

  • Compare results across multiple techniques (e.g., IF, WB, IP)

  • Consistent results across platforms increase confidence in specificity

What is the optimal protocol for conjugating an unconjugated ICE2 antibody with biotin in the laboratory?

For researchers needing to prepare their own biotin-conjugated ICE2 antibody, the following methodological approach is recommended:

• Material Selection:

  • Choose an appropriate biotinylation reagent (NHS-biotin, Sulfo-NHS-LC-biotin, or commercial conjugation kits)

  • Ensure antibody is in a compatible buffer (generally, phosphate or borate buffer without primary amines)

• Buffer Exchange Procedure (if needed):

  • Dialyze antibody against 0.1M sodium phosphate, 0.15M NaCl, pH 7.2-7.5

  • Alternatively, use desalting columns or centrifugal filters

  • Avoid buffers containing Tris, glycine, or other primary amines that interfere with conjugation

• Conjugation Protocol:

  • Prepare biotin reagent (typically 10-20 molar excess)

  • Add to antibody solution (1-5 mg/mL)

  • Incubate for 30 minutes to 2 hours at room temperature

  • For rapid conjugation, commercial kits offer protocols completing in less than 20 minutes with only 30 seconds hands-on time

• Purification Steps:

  • Remove excess biotin through dialysis or gel filtration

  • For column purification, use mobile phase of PBS or similar buffer

• Quality Control Assessment:

  • Determine biotin/protein ratio using HABA assay or mass spectrometry

  • Optimal labeling typically ranges from 3-8 biotin molecules per antibody

  • Verify activity through comparative ELISA against unconjugated antibody

What troubleshooting approaches address weak or inconsistent signals when using biotin-conjugated ICE2 antibody?

When experiencing suboptimal results with biotin-conjugated ICE2 antibody, consider the following systematic troubleshooting approaches:

• Signal Strength Optimization:

  • Titrate antibody concentration (typically 0.5-10 μg/mL range)

  • Extend primary antibody incubation time (overnight at 4°C versus 1-2 hours)

  • Try different detection systems (HRP-streptavidin, fluorescent-streptavidin)

  • Implement signal amplification methods (tyramide signal amplification or similar)

• Epitope Accessibility Enhancement:

  • Optimize antigen retrieval methods (heat-induced versus enzymatic)

  • Test multiple fixation protocols (paraformaldehyde, methanol, acetone)

  • Consider gentler cell/tissue permeabilization methods

  • For nuclear proteins like ICE2, ensure nuclear membrane is adequately permeabilized

• Background Reduction Strategies:

  • Include biotin blocking steps to reduce endogenous biotin signals

  • Implement more stringent washing protocols (increased duration, detergent concentration)

  • Pre-absorb detection reagents with sample matrix components

  • Use sample-specific blocking reagents (5% milk, BSA, normal serum)

• Antibody Stability Assessment:

  • Check for antibody degradation (run on SDS-PAGE)

  • Minimize freeze-thaw cycles (aliquot upon receipt)

  • Adhere to recommended storage conditions (-20°C or -80°C)

  • Consider adding stabilizing proteins (BSA) to diluted antibody

• Sample Preparation Considerations:

  • Verify protein expression level of ICE2 in your sample

  • Ensure samples were properly processed to preserve epitopes

  • Consider protein enrichment methods for low abundance targets

How can researchers differentiate between specific ICE2 signal and potential artifacts in immunostaining experiments?

Distinguishing genuine ICE2 signals from artifacts requires rigorous controls and careful interpretation:

• Essential Control Panel Development:

  • Negative controls: No primary antibody, isotype control, pre-immune serum

  • Positive controls: Tissues/cells with known ICE2 expression

  • Knockdown/knockout controls: siRNA-treated or CRISPR-modified samples

  • Competitive inhibition: Pre-incubation with immunizing peptide

• Expected Localization Pattern Analysis:

  • ICE2, as part of the Little Elongation Complex, should show predominantly nuclear localization with potential enrichment at transcriptionally active sites

  • Unexpected cytoplasmic staining may indicate cross-reactivity or experimental artifacts

  • Compare localization pattern with published literature on ICE2 distribution

• Multi-channel Imaging Correlation:

  • Co-stain with markers of transcriptionally active nuclear regions

  • Assess co-localization with other known LEC components

  • Use nuclear counterstains to confirm nuclear localization

• Signal Specificity Verification:

  • Signal should diminish in dose-dependent manner with competitive peptide

  • Pattern should be reproducible across multiple samples and experiments

  • Signal intensity should correlate with known expression levels across different cell types

What experimental design best addresses the dynamic interaction of ICE2 with other components of the Little Elongation Complex?

To study the dynamic interactions of ICE2 within the LEC, consider these methodological approaches:

• Co-immunoprecipitation Strategy:

  • Use biotin-conjugated ICE2 antibody with streptavidin beads for pull-down

  • Analyze co-precipitated proteins by mass spectrometry or Western blot

  • Include appropriate controls (IgG, beads only)

  • Consider crosslinking approaches to capture transient interactions

• Live-cell Imaging Approach:

  • Generate fluorescently tagged ICE2 and other LEC components

  • Employ fluorescence recovery after photobleaching (FRAP) to assess exchange dynamics

  • Use fluorescence resonance energy transfer (FRET) to measure direct protein-protein interactions

  • Analyze co-localization changes during transcriptional activation/repression

• Chromatin Dynamics Analysis:

  • Perform ChIP-seq for ICE2 and other LEC components

  • Identify regions of co-occupancy and exclusive binding

  • Correlate with RNA Pol II occupancy and transcriptional output

  • Study temporal dynamics following transcriptional stimulation

• Proximity-based Labeling Techniques:

  • Generate BioID or TurboID fusions with ICE2

  • Identify proteins in proximity to ICE2 under different cellular conditions

  • Compare results with known LEC components to identify novel interactions

  • Validate key interactions through orthogonal methods

• Structural Biology Integration:

  • Complement interaction studies with available structural data

  • Model interaction interfaces based on crosslinking mass spectrometry data

  • Validate models through mutagenesis of predicted interaction surfaces

How should expression levels of ICE2 be quantified across different experimental conditions?

Accurate quantification of ICE2 expression requires consideration of several methodological factors:

• Western Blot Quantification Protocol:

  • Use biotin-conjugated ICE2 antibody with streptavidin-HRP detection

  • Include loading controls (β-actin, GAPDH, total protein stain)

  • Generate standard curves with recombinant ICE2 for absolute quantification

  • Analyze using appropriate software (ImageJ, Image Lab) with background subtraction

• qRT-PCR Complementary Approach:

  • Design efficient primers spanning exon-exon junctions for ICE2 mRNA

  • Validate primer efficiency using standard curves

  • Select appropriate reference genes verified for stability under your experimental conditions

  • Calculate relative expression using 2^-ΔΔCt or standard curve methods

• Flow Cytometry Application:

  • Optimize cell fixation and permeabilization for nuclear protein detection

  • Use biotin-conjugated ICE2 antibody with fluorescent streptavidin

  • Include appropriate fluorescence-minus-one (FMO) controls

  • Report data as median fluorescence intensity (MFI)

• Immunohistochemistry Quantification Strategy:

  • Standardize staining protocol and imaging parameters

  • Use digital image analysis software for unbiased quantification

  • Score nuclear staining intensity and percent positive cells

  • Implement H-score or similar semi-quantitative approach

• Data Normalization Considerations:

  • Account for variations in cell number, protein content, or tissue area

  • Consider using ratio to housekeeping gene/protein expression

  • For tissue analyses, normalize to specific cell populations rather than total tissue

How does the performance of biotin-conjugated ICE2 antibody compare with other detection methods for studying the Little Elongation Complex?

Comparative analysis of different detection methods provides valuable insights for experimental design:

When designing experiments to study the Little Elongation Complex, researchers should consider combining complementary methods to overcome limitations of individual approaches.

What emerging technologies might enhance the application of ICE2 antibodies in transcriptional regulation research?

Several innovative technologies show promise for advancing ICE2 research:

• Single-cell Antibody-based Technologies:

  • Integration with single-cell RNA-seq for correlating ICE2 protein levels with transcriptomic profiles

  • Single-cell CyTOF for multi-parameter analysis of LEC components and associated factors

  • Spatial transcriptomics combined with ICE2 immunodetection for tissue-level analysis

• Super-resolution Microscopy Applications:

  • STORM/PALM microscopy for nanoscale localization of ICE2 within nuclear substructures

  • Expansion microscopy to physically enlarge samples for improved visualization of LEC components

  • Lattice light-sheet microscopy for dynamic tracking of ICE2 in living cells

• CRISPR-based Approaches:

  • CUT&RUN or CUT&Tag using ICE2 antibody for improved chromatin profiling

  • CRISPR activation/inhibition screens to identify regulators of ICE2 function

  • Endogenous tagging of ICE2 using CRISPR knock-in for physiological studies

• Multiplexed Antibody Methods:

  • Cyclic immunofluorescence for detecting dozens of proteins in the same sample

  • DNA-barcoded antibody methods for high-throughput protein quantification

  • Microfluidic antibody arrays for analyzing ICE2 interactions in limited samples

• Artificial Intelligence Integration:

  • Machine learning algorithms for identifying subtle patterns in ICE2 localization data

  • AI-assisted image analysis for quantifying complex co-localization patterns

  • Predictive modeling of ICE2 interactions based on multiple data types

These emerging technologies offer promising avenues for deeper understanding of ICE2's role in transcriptional regulation and the dynamics of the Little Elongation Complex.