ITCH (Ab-420) Antibody

What is ITCH (Ab-420) Antibody and what epitope does it recognize?

ITCH (Ab-420) Antibody is a rabbit polyclonal antibody that detects endogenous levels of total ITCH protein, a critical E3 ubiquitin-protein ligase in cellular signaling pathways. This antibody specifically recognizes the region surrounding the tyrosine 420 phosphorylation site (F-I-Y(p)-G-N) within human ITCH protein . The immunogen used to generate this antibody is a synthesized non-phosphopeptide derived from this specific region, enabling detection of the ITCH protein regardless of its phosphorylation status at this site . The antibody demonstrates reactivity to both human and mouse ITCH proteins, making it suitable for comparative studies across these species .

What are the validated applications for ITCH (Ab-420) Antibody?

Based on empirical validation data, ITCH (Ab-420) Antibody has been confirmed effective for the following applications:

• Western Blot (WB): Recommended dilutions range from 1:500-1:3000

• ELISA: Recommended dilutions range from 1:2000-1:10000

The antibody has been validated in Western blot analysis of extracts from mouse brain cells as well as HepG2 human liver cancer cell lines, demonstrating reliable detection of the target protein in diverse tissue and cell types . It's important to note that each new experimental system may require optimization of antibody concentration and assay conditions for optimal results.

What storage conditions maintain ITCH (Ab-420) Antibody stability?

For unconjugated ITCH (Ab-420) Antibody, optimal storage is at -20°C in its formulation buffer consisting of rabbit IgG in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, containing 150mM NaCl, 0.02% sodium azide and 50% glycerol . This formulation provides stability while preventing microbial contamination and freeze-thaw damage.

For conjugated versions of the antibody, storage recommendations vary by conjugate type. Fluorophore-conjugated antibodies (such as AF350, AF488, AF555) should generally be stored at 4°C in the dark for up to 6 months to prevent photobleaching and maintain signal intensity . Always refer to specific product documentation for conjugate-specific storage guidance.

How should ITCH (Ab-420) Antibody be validated for experimental specificity?

A multi-faceted validation approach is recommended:

• Peptide competition assay: As demonstrated in validation data, pre-incubating the antibody with the synthesized peptide immunogen effectively blocks specific binding in Western blot analysis of HepG2 cells . This approach confirms epitope-specific binding.

• Positive and negative control samples: Include known ITCH-expressing tissues (such as mouse brain) as positive controls and consider ITCH-knockout or knockdown samples as negative controls when available.

• Size verification: The ITCH protein has a calculated molecular weight of approximately 103 kDa . Verify that the detected band appears at the expected molecular weight.

• Cross-species comparison: The validated reactivity to both human and mouse samples provides an opportunity for specificity verification across species with conserved epitopes.

What are the optimal conditions for using ITCH (Ab-420) Antibody in complex tissue samples?

When working with complex tissue samples such as brain tissue:

• Sample preparation optimization: Thoroughly homogenize tissues in appropriate lysis buffers containing protease and phosphatase inhibitors to prevent degradation.

• Blocking optimization: Use 5% non-fat dry milk or BSA in TBST for Western blot applications to minimize background signal.

• Antibody dilution titration: Begin with the manufacturer's recommended range (1:500-1:3000 for WB) but perform a dilution series to determine optimal signal-to-noise ratio for your specific sample type.

• Incubation conditions: For brain tissue samples, overnight primary antibody incubation at 4°C often yields better results than shorter incubations at room temperature.

• Detection system selection: Choose detection systems compatible with the expected abundance of ITCH in your samples. Enhanced chemiluminescence (ECL) systems work well for moderate abundance proteins, while more sensitive detection methods may be required for low-abundance samples.

What considerations apply when using conjugated versions of ITCH (Ab-420) Antibody?

ITCH (Ab-420) Antibody is available in multiple conjugated forms with distinct spectral properties to support various experimental platforms:

When using conjugated antibodies:

• Protect from light during all handling steps

• Consider spectral overlap when designing multiplex experiments

• Include appropriate single-stained controls for compensation in flow cytometry

• Titrate antibody concentration for optimal signal-to-noise ratio

How can non-specific binding be reduced when using ITCH (Ab-420) Antibody?

If experiencing non-specific binding or high background:

• Increase blocking stringency: Extend blocking time to 2 hours or switch blocking reagent (BSA vs. milk) based on your detection system.

• Optimize antibody concentration: Further dilute the antibody beyond the recommended range if signal-to-noise ratio is poor.

• Add detergent: Increase Tween-20 concentration in wash buffers to 0.1-0.3% to reduce hydrophobic interactions.

• Pre-absorb the antibody: For tissues with known cross-reactivity issues, consider pre-absorbing the antibody with tissue lysate from a negative control sample.

• Peptide competition: Use the immunizing peptide as a competitive inhibitor to distinguish between specific and non-specific binding.

What are the key considerations for quantitative analysis of ITCH expression using this antibody?

For reliable quantitative analysis:

• Loading control selection: Choose appropriate loading controls based on your experimental system (β-actin, GAPDH, or total protein staining).

• Linear dynamic range determination: Perform a sample dilution series to ensure quantification occurs within the linear detection range of both the antibody and detection system.

• Normalization strategy: For phosphorylation studies, normalize phospho-specific signals to total ITCH protein levels rather than just to loading controls.

• Technical replicates: Include at least three technical replicates for statistical validity.

• Image acquisition parameters: Use identical exposure settings when comparing samples across different blots or immunofluorescence images.

How can ITCH (Ab-420) Antibody be applied to study ITCH's role in protein ubiquitination?

ITCH functions as an E3 ubiquitin ligase involved in protein degradation pathways. To study this function:

• Co-immunoprecipitation studies: Use ITCH (Ab-420) Antibody to pull down ITCH complexes and analyze associated proteins to identify novel substrates.

• Ubiquitination assays: Combine with anti-ubiquitin antibodies in Western blot analyses to detect ubiquitinated substrates after ITCH immunoprecipitation.

• Inhibitor studies: Examine changes in ITCH-substrate interactions following treatment with proteasome inhibitors (MG132) or deubiquitinase inhibitors.

• Phosphorylation impact: Since the antibody targets a region near Tyr420, it can be used alongside phospho-specific antibodies to determine how phosphorylation affects ITCH's ubiquitin ligase activity.

• Degradation kinetics: Track substrate protein levels over time following ITCH activation or inhibition to characterize degradation kinetics.

What approaches are recommended for studying ITCH protein interactions with its known binding partners?

To investigate protein-protein interactions:

• Proximity ligation assay (PLA): Combine ITCH (Ab-420) Antibody with antibodies against suspected binding partners to visualize and quantify interactions in situ.

• FRET analysis: Use fluorophore-conjugated versions of ITCH (Ab-420) Antibody in combination with differently-labeled partner antibodies to study interaction dynamics in living cells.

• Pull-down confirmation: Follow up on observed interactions with reciprocal immunoprecipitation using both ITCH (Ab-420) Antibody and antibodies against binding partners.

• Domain mapping: Combine with deletion mutant studies to identify specific interaction domains.

• Stimulus-response experiments: Monitor how interactions change in response to cellular stressors, signaling activators, or inhibitors.

How does ITCH (Ab-420) Antibody compare to antibodies targeting other epitopes of ITCH protein?

When selecting between different ITCH antibodies:

• Epitope accessibility: The Ab-420 antibody targets a region around tyrosine 420, which may have different accessibility depending on protein conformation and interaction partners compared to antibodies targeting N-terminal or C-terminal epitopes.

• Post-translational modification sensitivity: Unlike phospho-specific antibodies, ITCH (Ab-420) Antibody detects total ITCH protein regardless of phosphorylation status, making it suitable for normalization in phosphorylation studies.

• Cross-reactivity profile: The specificity for human and mouse ITCH proteins makes this antibody suitable for comparative studies across these species, but researchers working with other model organisms should verify cross-reactivity.

• Application versatility: The validated applications (WB, ELISA) may differ from other epitope-targeting antibodies that might be validated for additional applications like immunohistochemistry or immunofluorescence.

• Isoform detection: Consider whether the targeted epitope is present in all known ITCH isoforms relevant to your research question.

What experimental controls should be included when using ITCH (Ab-420) Antibody in new research contexts?

For robust experimental design:

• Technical validation controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Peptide competition control using the immunizing peptide

• Biological controls:

  • Positive control tissues/cells with known ITCH expression (mouse brain, HepG2 cells)

  • ITCH knockdown/knockout validation where available

  • Cross-species comparison when studying conserved mechanisms

• Quantification controls:

  • Standard curve for quantitative applications

  • Loading controls appropriate to your experimental system

  • Replicate samples for statistical analysis