Cleaved-ITIH4 (R661) Antibody

What is Cleaved-ITIH4 (R661) Antibody and what epitope does it recognize?

The Cleaved-ITIH4 (R661) Antibody is a rabbit polyclonal antibody that specifically detects endogenous levels of activated ITI-H4 70k protein fragments resulting from cleavage adjacent to arginine 661 (R661) . The antibody was raised against a synthesized peptide derived from human ITIH4, specifically targeting the amino acid range 612-661 . This specificity makes it valuable for detecting the cleaved form of ITIH4 rather than the intact protein, allowing researchers to monitor ITIH4 processing in various biological contexts.

What is the biological significance of ITIH4 protein and its cleavage?

ITIH4 functions as a Type II acute-phase protein involved in inflammatory responses to trauma and may play a role in liver development or regeneration . Recent research has revealed that ITIH4 acts as a protease inhibitor through a novel inhibitory mechanism . The protein is cleaved by several human proteases within a protease-susceptible region, enabling it to function as a protease inhibitor for key enzymes involved in intravascular host defense, including mannan-binding lectin–associated serine protease-1 (MASP-1), MASP-2, and plasma kallikrein . This cleavage-dependent inhibitory function represents a previously unrecognized biological role for ITIH4.

What are the recommended applications for Cleaved-ITIH4 (R661) Antibody?

The Cleaved-ITIH4 (R661) Antibody is suitable for multiple research applications:

• Immunohistochemistry (IHC): Recommended dilution range of 1:100-1:300

• Immunofluorescence (IF): Recommended dilution range of 1:50-200

• Enzyme-Linked Immunosorbent Assay (ELISA): Recommended dilution of 1:10000

For optimal results in IHC applications, researchers should test different dilutions within the recommended range to determine the optimal concentration for their specific tissue samples and experimental conditions.

How is ITIH4 processed in the body and what fragments are generated?

ITIH4 undergoes complex proteolytic processing in vivo:

• Initial cleavage by plasma kallikrein generates 100 kDa and 35 kDa fragments

• The 100 kDa fragment is further converted to a 70 kDa fragment

• When incubated with MASP-1, ITIH4 is cleaved into 80 kDa and 42 kDa fragments, with the 42 kDa fragment further processed into a 37 kDa fragment

• MASP-2 cleaves ITIH4 slightly faster than MASP-1, producing an 85 kDa fragment that is subsequently processed into 80 kDa and 37 kDa fragments

• After extended incubation (24 hours), both MASP-1 and MASP-2 generate a 60 kDa fragment through secondary cleavage of the 80 kDa fragment

These distinct fragmentation patterns can help researchers identify which proteases are active in their biological samples.

What is the recommended methodology for validating the specificity of Cleaved-ITIH4 (R661) Antibody?

To validate the specificity of the Cleaved-ITIH4 (R661) Antibody in your experiments, consider the following methodological approach:

• Positive control: Include samples with known ITIH4 cleavage (e.g., serum samples incubated with MASP-1 or MASP-2)

• Negative control: Use samples with intact ITIH4 or samples treated with protease inhibitors to prevent ITIH4 cleavage

• Peptide competition assay: Pre-incubate the antibody with the immunogenic peptide (aa 612-661) before application to samples

• Western blot analysis: Confirm the detection of appropriately sized fragments (70 kDa) corresponding to cleaved ITIH4

• Comparison with alternative antibodies: If available, compare results with other antibodies against different ITIH4 epitopes

This multi-faceted validation approach will help establish confidence in the specificity of the antibody for cleaved ITIH4.

What post-translational modifications occur in ITIH4 and how might they affect antibody recognition?

ITIH4 undergoes several post-translational modifications that may impact antibody binding:

• N-glycosylation is present in plasma ITIH4

• O-glycosylation occurs in urinary ITIH4, particularly on threonine residues in the region from Thr-719 to Thr-725, consisting primarily of core 1 or possibly core 8 glycans (mainly Hex(HexNAc)(2), but also some Hex(3)(HexNAc)(3))

• Interestingly, ITIH4 is N-glycosylated but not O-glycosylated in plasma

These modifications may affect antibody recognition if they occur near or within the epitope region (aa 612-661). Researchers should be aware that sample preparation methods that alter glycosylation patterns might affect antibody binding efficiency. For consistent results, standardized sample preparation protocols are recommended.

What are the appropriate storage and handling conditions for Cleaved-ITIH4 (R661) Antibody?

For optimal performance and longevity, the Cleaved-ITIH4 (R661) Antibody should be:

• Stored at -20°C for up to 1 year from the date of receipt

• Avoid repeated freeze-thaw cycles to maintain antibody activity

• The antibody is typically formulated in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• When handling, wear appropriate personal protective equipment due to the presence of sodium azide

• For short-term use, small aliquots can be maintained at 4°C for up to one week

Following these storage recommendations will help maintain antibody performance and extend its usable lifespan in the laboratory.

How can Cleaved-ITIH4 (R661) Antibody be used to investigate ITIH4's role as a protease inhibitor?

To investigate ITIH4's novel function as a protease inhibitor, researchers can employ the following methodological approaches using the Cleaved-ITIH4 (R661) Antibody:

• Comparative analysis of ITIH4 cleavage patterns in response to different proteases:

  • Incubate recombinant ITIH4 with various proteases (MASP-1, MASP-2, kallikrein, MMP-7, MMP-13, thrombin, papain, plasmin)

  • Use Western blotting with Cleaved-ITIH4 (R661) Antibody to detect and compare resulting fragment patterns

  • Quantify the relationship between ITIH4 cleavage and inhibition of protease activity

• Immunoprecipitation studies to identify ITIH4-protease complexes:

  • Use the antibody to isolate cleaved ITIH4-protease complexes from biological samples

  • Characterize the composition and stability of these complexes using mass spectrometry

• Time-course experiments to monitor ITIH4 cleavage kinetics:

  • Follow the appearance of cleaved ITIH4 over time in response to protease exposure

  • Correlate cleavage patterns with changes in protease activity to establish temporal relationships

This multi-parameter approach will provide insights into the mechanism by which ITIH4 functions as a protease inhibitor following its cleavage.

What are the technical challenges in detecting ITIH4 cleavage in complex biological samples?

Detecting ITIH4 cleavage in complex biological samples presents several technical challenges:

• Distinguishing specific cleavage fragments:

  • Multiple proteases can cleave ITIH4, generating fragments of similar molecular weights

  • The Cleaved-ITIH4 (R661) Antibody detects only fragments resulting from cleavage adjacent to R661

  • Consider using additional antibodies targeting different cleavage sites for comprehensive analysis

• Background issues in immunohistochemistry:

  • Non-specific binding may occur in tissue samples with high protease activity

  • Optimize blocking conditions (use 5-10% normal serum from the same species as the secondary antibody)

  • Include appropriate negative controls (primary antibody omission, non-immune IgG substitution)

• Temporal dynamics of ITIH4 cleavage:

  • ITIH4 undergoes rapid and sequential cleavage events

  • Sample collection timing is critical for capturing specific cleavage intermediates

  • Consider using protease inhibitor cocktails immediately after sample collection to "freeze" the cleavage state

• Post-translational modifications affecting epitope recognition:

  • Develop sample preparation protocols that preserve the native state of the R661 cleavage site

  • Be aware that urinary and plasma ITIH4 may have different glycosylation patterns

Addressing these challenges requires careful experimental design and validation of detection methods in each specific biological context.

How can Cleaved-ITIH4 (R661) Antibody be used to develop biomarker assays for inflammatory conditions?

The Cleaved-ITIH4 (R661) Antibody can be leveraged to develop biomarker assays for inflammatory conditions through the following methodological approach:

• Sandwich ELISA development:

  • Use Cleaved-ITIH4 (R661) Antibody as a capture antibody

  • Pair with a detection antibody against a different ITIH4 epitope

  • Establish standard curves using recombinant cleaved ITIH4 fragments

  • Validate assay sensitivity and specificity in clinical samples

• Multiplex immunoassay integration:

  • Incorporate the antibody into bead-based multiplex platforms

  • Combine with markers of inflammation (cytokines, acute phase proteins)

  • Develop algorithms correlating ITIH4 cleavage patterns with disease progression

• Tissue microarray analysis:

  • Apply optimized IHC protocols to tissue microarrays from patients with inflammatory conditions

  • Quantify cleaved ITIH4 levels using digital pathology approaches

  • Correlate expression patterns with clinical parameters and outcomes

• Validation using complementary techniques:

  • Confirm ELISA/IHC findings with Western blot analysis of patient samples

  • Use mass spectrometry to validate specific ITIH4 cleavage products

This comprehensive approach will help establish whether cleaved ITIH4 has value as a biomarker for specific inflammatory conditions.

What is the relationship between ITIH4 cleavage and the lectin pathway of complement activation?

The relationship between ITIH4 cleavage and the lectin pathway of complement activation involves a complex interplay that can be studied using the Cleaved-ITIH4 (R661) Antibody:

• MASP-mediated ITIH4 cleavage:

  • MASP-1 and MASP-2, key proteases in the lectin pathway, efficiently cleave ITIH4

  • PRM (pattern recognition molecule)-bound MASP-1 cleaves ITIH4, indicating relevance during lectin pathway activation

  • MASP-3, another protease associated with the lectin pathway, does not cleave ITIH4

• Inhibitory feedback mechanism:

  • Cleaved ITIH4 forms a noncovalent, inhibitory complex with the executing protease

  • This complex depends on the ITIH4 von Willebrand factor A domain

  • ITIH4 inhibits MASPs by sterically preventing larger protein substrates from accessing their active sites

• Experimental approach to study this relationship:

  • Immobilize mannose-binding lectin (MBL) on a mannose surface

  • Add cleaved MASP-1 to form ligand-bound MBL-MASP-1 complexes

  • Incubate with ITIH4 and monitor cleavage patterns over time

  • Use Cleaved-ITIH4 (R661) Antibody to detect formed complexes via immunoassays

Understanding this relationship may provide insights into novel regulatory mechanisms of complement activation during inflammatory responses.

What controls should be included when using Cleaved-ITIH4 (R661) Antibody in immunohistochemistry?

When designing immunohistochemistry experiments with the Cleaved-ITIH4 (R661) Antibody, the following controls should be included:

• Positive tissue controls:

  • Liver tissue (ITIH4 is primarily expressed in the liver)

  • Tissues from inflammatory sites (where proteases that cleave ITIH4 are likely active)

• Negative controls:

  • Primary antibody omission: Replace primary antibody with antibody diluent

  • Isotype control: Use non-immune rabbit IgG at the same concentration

  • Peptide competition: Pre-absorb antibody with immunizing peptide (aa 612-661)

• Processing controls:

  • Compare fresh vs. fixed tissues to assess epitope sensitivity to fixation

  • Test multiple antigen retrieval methods to optimize signal

  • Include tissues treated with protease inhibitors to prevent ITIH4 cleavage

• Interpretation controls:

  • Stain serial sections with antibodies against total ITIH4 to compare with cleaved-specific staining

  • Include tissues from ITIH4-knockout models if available

This comprehensive control strategy will ensure reliable and interpretable immunohistochemistry results.

How should researchers optimize Western blot protocols for detecting cleaved ITIH4 fragments?

Optimizing Western blot protocols for detecting cleaved ITIH4 fragments requires several methodological considerations:

• Sample preparation:

  • Use fresh samples or add protease inhibitors immediately after collection

  • For serum/plasma samples, consider depleting abundant proteins to enrich for ITIH4

  • Include positive controls (e.g., ITIH4 incubated with MASP-1 or MASP-2)

• Gel selection and running conditions:

  • Use gradient gels (4-15%) to resolve the full range of ITIH4 fragments (35-120 kDa)

  • Run at lower voltage (80-100V) to improve resolution of closely sized fragments

  • Consider using Phos-tag gels if phosphorylation affects cleavage patterns

• Transfer optimization:

  • Use wet transfer for large proteins (intact ITIH4 ~120 kDa)

  • Extend transfer time for larger fragments (2-3 hours or overnight at 4°C)

  • Add 0.1% SDS to transfer buffer to improve transfer of larger fragments

• Antibody conditions:

  • Optimize antibody dilution (start with 1:1000 and adjust as needed)

  • Extended primary antibody incubation (overnight at 4°C)

  • Use highly sensitive detection systems (ECL-Prime or fluorescent secondary antibodies)

• Data analysis:

  • Document all visible bands and compare to expected fragment sizes:

    • Intact ITIH4: ~120 kDa

    • Major cleaved fragments: 80-85 kDa, 70 kDa, 60 kDa, 42 kDa, 37 kDa

This optimized protocol will enable reliable detection and characterization of ITIH4 cleavage products in various experimental systems.

How can Cleaved-ITIH4 (R661) Antibody contribute to understanding disease mechanisms?

The Cleaved-ITIH4 (R661) Antibody offers valuable opportunities for investigating disease mechanisms through the following research applications:

• Inflammatory disease models:

  • ITIH4 functions as a Type II acute-phase protein involved in inflammatory responses

  • The antibody can track ITIH4 processing during different phases of inflammation

  • Correlate ITIH4 cleavage patterns with disease progression and severity

• Protease dysregulation studies:

  • ITIH4 is cleaved by various proteases including MASP-1, MASP-2, MMP-7, MMP-13, thrombin, papain, plasmin, and kallikrein

  • The antibody can assess protease activity indirectly through ITIH4 cleavage patterns

  • Identify disease-specific ITIH4 fragmentation profiles

• Liver pathophysiology:

  • ITIH4 may play a role in liver development or regeneration

  • The antibody can monitor changes in ITIH4 processing during liver injury and repair

  • Investigate the relationship between ITIH4 cleavage and hepatic acute-phase response

• Complement activation disorders:

  • ITIH4 inhibits key proteases in the lectin pathway of complement activation

  • The antibody can assess whether aberrant ITIH4 processing contributes to dysregulated complement activation

  • Explore therapeutic potential of modulating ITIH4-protease interactions

This research may ultimately reveal new diagnostic biomarkers and therapeutic targets for conditions involving dysregulated protease activity and inflammation.

What methodological approaches can be used to quantify ITIH4 cleavage in clinical samples?

Quantifying ITIH4 cleavage in clinical samples requires robust methodological approaches:

• Quantitative sandwich ELISA:

  • Capture antibody: Anti-total ITIH4

  • Detection antibody: Cleaved-ITIH4 (R661) Antibody

  • Standard curve: Recombinant cleaved ITIH4 fragments

  • Sample types: Serum, plasma, or tissue homogenates

  • Detection limit: Establish with serial dilutions of standards

• Multiplex bead-based immunoassay:

  • Couple Cleaved-ITIH4 (R661) Antibody to microspheres

  • Analyze using flow cytometry-based platforms

  • Advantage: Simultaneous measurement of multiple analytes

  • Normalization: Include total ITIH4 measurement in the panel

• Digital pathology quantification:

  • Apply validated IHC protocol with Cleaved-ITIH4 (R661) Antibody

  • Use digital image analysis software to quantify:

    • Staining intensity

    • Percentage of positive cells

    • Tissue distribution patterns

  • Standardize with calibration slides

• Mass spectrometry-based approaches:

  • Immunoprecipitate with Cleaved-ITIH4 (R661) Antibody

  • Analyze by LC-MS/MS to identify and quantify specific fragments

  • Advantage: Provides exact cleavage site information

  • Challenge: Requires specialized equipment and expertise

Each approach has strengths and limitations, and the optimal method depends on the specific research question and available clinical samples.

What recent technological advances could enhance research using Cleaved-ITIH4 (R661) Antibody?

Several technological advances can enhance research using the Cleaved-ITIH4 (R661) Antibody:

• Single-cell proteomics:

  • Combine the antibody with mass cytometry (CyTOF) for single-cell analysis

  • Investigate cell-specific responses to proteases that cleave ITIH4

  • Correlate with other protease substrates at single-cell resolution

• Proximity ligation assays (PLA):

  • Pair Cleaved-ITIH4 (R661) Antibody with antibodies against potential binding partners

  • Visualize protein-protein interactions involving cleaved ITIH4 in situ

  • Quantify the spatial distribution of these interactions in tissues

• Tissue clearing and 3D imaging:

  • Apply the antibody to cleared tissue samples for whole-organ imaging

  • Visualize the three-dimensional distribution of cleaved ITIH4

  • Correlate with vascular networks and inflammatory foci

• CRISPR-Cas9 gene editing:

  • Generate cell lines or animal models with modified ITIH4 cleavage sites

  • Use the antibody to validate the functional consequences of these modifications

  • Investigate structure-function relationships in ITIH4's inhibitory mechanism

• Microfluidic platforms:

  • Develop chip-based assays incorporating the antibody

  • Enable real-time monitoring of ITIH4 cleavage in flowing biological fluids

  • Potential application in point-of-care diagnostics

These technological advances provide new opportunities to investigate the complex biology of ITIH4 processing and its implications in health and disease.