Cleaved-CD97 (L530) Antibody

What is the Cleaved-CD97 (L530) Antibody and what epitope does it recognize?

The Cleaved-CD97 (L530) Antibody is a polyclonal antibody that specifically recognizes the cleaved form of CD97 Antigen Subunit Alpha at leucine 530 position. This antibody detects endogenous levels of activated CD97 Alpha protein resulting from cleavage adjacent to L530. The antibody is typically produced against synthesized peptides derived from the internal region of human CD97 alpha, specifically from amino acids 481-530 . The specificity for the cleaved form makes this antibody particularly valuable for studying CD97 processing and activation mechanisms.

What are the recommended applications for Cleaved-CD97 (L530) Antibody?

Based on validation data, Cleaved-CD97 (L530) Antibody is suitable for Western Blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications . For Western Blotting, the recommended dilution ranges from 1:500 to 1:3000, while for ELISA, dilutions of 1:1000 to 1:5000 are suggested . These applications are particularly useful for detecting cleaved CD97 in cell lysates and tissue samples, making the antibody valuable for investigating CD97 processing in various experimental conditions.

What species reactivity has been confirmed for commercially available Cleaved-CD97 (L530) Antibodies?

The available Cleaved-CD97 (L530) Antibodies demonstrate varying reactivity profiles. Some antibodies show reactivity primarily with human samples , while others have been validated for human, rat, and mouse samples . This cross-species reactivity profile is particularly important when designing comparative studies across different animal models. Researchers should confirm the specific reactivity of their chosen antibody before conducting cross-species studies.

What is known about the structure and processing of CD97 that makes the cleaved form significant?

CD97 undergoes autoproteolytic cleavage at its GPS (G-protein coupled receptor proteolysis site), generating a membrane-bound C-terminal fragment (CTF) and an extracellular N-terminal fragment (NTF) . This cleavage is critical for CD97 function. Research has demonstrated that in glioblastoma cells, the majority of CD97 receptors undergo cleavage regardless of isoform expression . Additionally, the CD97 NTF undergoes glycosylation, which affects its apparent molecular weight in experimental assays . The cleaved form at L530 is particularly significant as it represents the activated state of CD97 that participates in downstream signaling pathways.

How can Cleaved-CD97 (L530) Antibody be utilized to study CD97 signaling pathways in cancer models?

The Cleaved-CD97 (L530) Antibody serves as a crucial tool for studying CD97's role in cancer signaling networks, particularly in glioblastoma where CD97 promotes glycolytic metabolism via the MAPK pathway . Researchers can employ this antibody in combination with phospho-specific antibodies to examine how CD97 cleavage correlates with downstream MAPK activation. Experiments should include time-course analyses following stimulation with potential CD97 ligands, such as THY1/CD90 which has been identified as a likely ligand in glioblastoma . Additionally, co-immunoprecipitation studies using this antibody can help identify binding partners specific to the cleaved form, providing insights into differential recruitment of signaling mediators like β-arrestin, which depends on phosphorylation of CD97's C-terminus .

What methodological considerations are important when using Cleaved-CD97 (L530) Antibody to differentiate between cleaved and uncleaved forms in experimental settings?

When designing experiments to distinguish between cleaved and uncleaved CD97 forms, several methodological considerations are critical:

• Sample preparation: Use phosphatase inhibitors in lysis buffers to preserve phosphorylation-dependent signals related to CD97 activation.

• Control samples: Include CD97 S531A mutant (non-cleavable) as a negative control .

• Deglycosylation treatment: Consider enzymatic deglycosylation of samples before Western blotting to resolve discrepancies in the expected molecular weights of the NTF .

• Isoform-specific analysis: Design primers for RT-PCR to distinguish between CD97 isoforms with different numbers of EGF repeats, as shorter isoforms (3-4 EGF repeats) appear to be predominant in glioblastoma .

• Dual staining approach: Use antibodies against both ECD and ICD of CD97 to comprehensively track cleavage products .

This methodological approach enables accurate discrimination between processing states of CD97 in complex biological samples.

How can researchers optimize Western blot protocols specifically for detecting cleaved CD97 (L530)?

Optimizing Western blot protocols for cleaved CD97 (L530) detection requires special considerations:

Additionally, researchers should consider running gradient gels (4-15%) to better resolve both the cleaved fragments and full-length protein simultaneously. For challenging samples with low expression, signal enhancement systems compatible with polyclonal rabbit antibodies may improve detection sensitivity.

What experimental approaches can confirm the specificity of Cleaved-CD97 (L530) Antibody in functional studies?

To validate antibody specificity for functional studies, researchers should implement a multi-faceted approach:

• Genetic validation: Compare staining patterns between wild-type cells and CD97 knockout/knockdown models .

• Peptide competition assays: Pre-incubate the antibody with excess immunizing peptide (amino acids 481-530) to confirm signal specificity .

• Non-cleavable mutant controls: Use the CD97 S531A mutant as a negative control in parallel experiments .

• Cross-antibody validation: Compare results with other antibodies targeting different epitopes of CD97.

• Recombinant protein controls: Use purified cleaved and uncleaved CD97 proteins as positive and negative controls.

These validation steps are essential before proceeding to functional studies, particularly when investigating CD97's role in promoting glycolytic metabolism or MAPK pathway activation in cancer models .

How does CD97 cleavage at L530 relate to its function in tumor metabolism and signaling?

CD97 cleavage at L530 represents a critical regulatory event with significant implications for tumor metabolism. Research demonstrates that cleaved CD97 promotes glycolytic metabolism via the MAPK pathway in glioblastoma . The cleavage event appears to be necessary for proper receptor activation and subsequent recruitment of signaling mediators like β-arrestin, which depends on phosphorylation of CD97's C-terminus .

The metabolic functions regulated by cleaved CD97 are particularly relevant in cancer research as they support the Warburg effect - the preferential use of glycolysis even in the presence of oxygen. This metabolic reprogramming provides cancer cells with building blocks for rapid proliferation. Detecting the cleaved form using the L530 antibody allows researchers to specifically monitor the activated state of CD97 that drives these metabolic changes, providing a more precise biomarker for oncogenic activity than total CD97 levels.

What is the significance of CD97 isoform variation in research using Cleaved-CD97 (L530) Antibody?

CD97 exists in multiple isoforms with varying numbers of EGF repeats in the extracellular domain. Research indicates that shorter isoforms (with three or four EGF repeats) predominate in glioblastoma . This isoform variation has important implications for research using the Cleaved-CD97 (L530) Antibody:

• Molecular weight interpretation: Different isoforms yield cleaved fragments of varying sizes, particularly for the NTF.

• Glycosylation patterns: The extent of glycosylation can differ between isoforms, affecting antibody recognition and apparent molecular weights .

• Ligand specificity: Different isoforms may preferentially interact with specific ligands (e.g., THY1/CD90 in glioblastoma) .

• Experimental design: Researchers should consider isolating and analyzing specific isoforms when studying cleaved CD97 functions.

When using the Cleaved-CD97 (L530) Antibody, it's essential to note the specific isoform being studied and consider how this might influence experimental outcomes and interpretations, particularly in comparative studies across different tissue types.

How can Cleaved-CD97 (L530) Antibody be employed in studies examining potential therapeutic targeting of CD97?

The Cleaved-CD97 (L530) Antibody represents a valuable tool for therapeutic development targeting CD97, particularly in glioblastoma research. Several strategic applications include:

• Target validation: Using the antibody to confirm CD97 cleavage status in patient-derived samples can help identify populations likely to respond to CD97-targeted therapies.

• Mechanism studies: The antibody can help elucidate how CD97 cleavage affects downstream MAPK signaling and metabolism , informing combination therapy approaches.

• Antibody-drug conjugate development: Research has demonstrated that CD97-targeted antibody-drug conjugates (ADCs) can selectively kill tumor cells in vitro . The Cleaved-CD97 (L530) Antibody can be used to validate specificity of ADCs for the cleaved form.

• Therapeutic resistance monitoring: Tracking changes in CD97 cleavage patterns during treatment can potentially identify mechanisms of therapy resistance.

• Biomarker development: The antibody can help establish whether cleaved CD97 levels correlate with disease progression or treatment response.

These applications highlight the translational potential of research utilizing this antibody beyond basic mechanistic studies.

What are the most common technical challenges when working with Cleaved-CD97 (L530) Antibody and how can they be addressed?

Researchers working with Cleaved-CD97 (L530) Antibody frequently encounter several technical challenges:

Additionally, using phosphatase inhibitors in sample preparation is critical when studying CD97 signaling, as the phosphorylation of CD97's C-terminus is necessary for β-arrestin recruitment and downstream signaling .

How should researchers interpret ambiguous molecular weight patterns when using Cleaved-CD97 (L530) Antibody?

Interpreting molecular weight patterns for CD97 can be challenging due to multiple factors. Researchers should consider:

• Post-translational modifications: CD97 NTF undergoes extensive glycosylation, resulting in higher apparent molecular weights than predicted . Deglycosylation treatment resolves these discrepancies.

• Isoform variation: CD97 exists in multiple isoforms with varying numbers of EGF repeats, affecting fragment sizes .

• Complete vs. partial cleavage: Some experimental conditions may result in a mixture of cleaved and uncleaved forms.

• Degradation products: Additional bands may represent proteolytic degradation during sample preparation.

To accurately interpret results:

• Include both glycosylated and deglycosylated controls

• Run known CD97 isoform standards for size comparison

• Use antibodies against both the ECD and ICD to track all fragments

• Include the non-cleavable S531A mutant as a control for uncleaved CD97

This comprehensive approach allows for reliable identification of specific CD97 forms in complex samples.

What storage and handling precautions are critical for maintaining Cleaved-CD97 (L530) Antibody functionality?

Proper storage and handling of Cleaved-CD97 (L530) Antibody is essential for maintaining its functionality over time:

• Storage temperature: Store at -20°C for up to 1 year from the date of receipt .

• Aliquoting: Divide the antibody into small working aliquots to avoid repeated freeze-thaw cycles .

• Buffer composition: The antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide , which helps maintain stability.

• Thawing procedure: Thaw aliquots on ice or at 4°C rather than at room temperature.

• Working dilution preparation: Prepare fresh working dilutions on the day of use and discard unused diluted antibody.

• Shipping considerations: The antibody is typically shipped with ice packs and should be transferred to -20°C storage immediately upon receipt.

Following these guidelines will help ensure consistent antibody performance across experiments and extend the usable lifespan of this research tool.

How can Cleaved-CD97 (L530) Antibody contribute to understanding CD97's role in the tumor microenvironment?

The Cleaved-CD97 (L530) Antibody offers valuable insights into CD97's role in tumor-microenvironment interactions. CD97 has been implicated in leukocyte migration and plays an essential role in immune cell functions . Researchers can use this antibody to:

• Map the distribution of cleaved CD97 within the tumor microenvironment through immunohistochemistry or immunofluorescence microscopy.

• Investigate CD97-mediated interactions between tumor cells and stromal components, particularly with THY1/CD90-expressing cells .

• Study how CD97 cleavage status affects immune cell recruitment and function within tumors.

• Examine the relationship between CD97 processing and extracellular matrix remodeling in invasion processes.

• Analyze how hypoxic or acidic microenvironments influence CD97 cleavage patterns.

These applications provide critical insights into how CD97 processing contributes to tumor progression beyond cell-autonomous effects, potentially revealing new therapeutic strategies targeting the tumor microenvironment.

What advanced imaging techniques can be combined with Cleaved-CD97 (L530) Antibody for spatial analysis of CD97 processing?

Integrating Cleaved-CD97 (L530) Antibody with advanced imaging methodologies enables sophisticated spatial analysis of CD97 processing:

• Super-resolution microscopy (STORM, PALM): Allows nanoscale visualization of cleaved CD97 distribution in membrane microdomains.

• Proximity ligation assay (PLA): Detects interactions between cleaved CD97 and potential binding partners like THY1/CD90 .

• Multiplexed immunofluorescence: Simultaneously visualizes cleaved CD97 alongside MAPK pathway components and metabolic markers.

• Live-cell FRET biosensors: When combined with fluorescently tagged binding partners, monitors real-time CD97 cleavage dynamics.

• CODEX or Imaging Mass Cytometry: Enables highly multiplexed spatial profiling of cleaved CD97 in relation to dozens of other markers in tissue sections.

These technologies help resolve how CD97 cleavage status correlates with specific subcellular localizations and protein-protein interactions in situ, providing deeper insights into the spatial regulation of CD97 signaling and its functional consequences.

How might Cleaved-CD97 (L530) Antibody be utilized in developing personalized medicine approaches for glioblastoma?

The Cleaved-CD97 (L530) Antibody has significant potential in advancing personalized medicine for glioblastoma through multiple applications:

• Patient stratification: Immunohistochemical analysis of patient biopsies using this antibody can identify tumors with high levels of cleaved CD97, potentially identifying patients who might benefit from CD97-targeted therapies .

• Therapeutic monitoring: Serial liquid biopsies analyzed for cleaved CD97 fragments could serve as a biomarker for treatment response.

• Ex vivo drug sensitivity testing: Patient-derived organoids or explants can be assessed for CD97 cleavage status before and after experimental treatments.

• Companion diagnostic development: The antibody could form the basis of companion diagnostic assays for future CD97-targeted therapeutics.

• Combination therapy rationale: By identifying patients with active CD97-MAPK signaling , the antibody can help guide rational combinations with other pathway inhibitors.

Given that CD97 (ADGRE5) shows de novo expression in GBM compared to healthy brain tissue , therapeutics targeting this pathway may offer improved specificity with reduced off-target effects on normal brain tissue.

By implementing these advanced research applications, scientists can leverage the Cleaved-CD97 (L530) Antibody to gain deeper insights into CD97 biology and develop more effective, personalized treatment strategies for challenging malignancies like glioblastoma.