Cleaved-ITGA7 (E959) Antibody

What is Cleaved-ITGA7 (E959) Antibody and what epitope does it recognize?

Cleaved-ITGA7 (E959) polyclonal antibody specifically recognizes the Cleaved-ITGA7 at the E959 cleavage site. This antibody binds to endogenous Integrin Alpha-7 that has been cleaved into Integrin Alpha-7 Heavy Chain, Integrin Alpha-7 Light Chain, and the Integrin Alpha-7 70 kDa form. The antibody is generated against a synthesized peptide derived from the human ITGA7 protein at the amino acid range 940-989, with the immunogen region specifically covering the 910-990 C-terminal domain . This specificity makes it valuable for detecting post-translationally modified forms of ITGA7, particularly after proteolytic processing events.

What are the recommended applications for Cleaved-ITGA7 (E959) Antibody?

The Cleaved-ITGA7 (E959) polyclonal antibody has been validated for use in Western Blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) research applications . For Western blotting, the recommended dilution range is 1:500-1:2000, while for ELISA the recommended dilution is significantly higher at 1:40000 . The antibody has demonstrated specific reactivity against human and monkey samples, making it suitable for comparative studies across these species . Unlike some other ITGA7 antibodies that recognize different domains (such as the extracellular domain recognized by antibody clone 9.1), the E959 antibody specifically targets the cleaved form, providing unique research capabilities .

What is the molecular basis of ITGA7 and its physiological significance?

The ITGA7 gene encodes integrin subunit alpha 7, which belongs to the integrin alpha chain family . Integrins function as heterodimeric integral membrane proteins composed of alpha and beta chains that mediate cell-cell and cell-matrix interactions . The alpha 7/beta-1 integrin serves as the primary laminin-1 receptor and is predominantly expressed in skeletal and cardiac muscle tissues, as well as in certain tumor cells . The full-length ITGA7 protein has a molecular weight of approximately 128.9 kilodaltons , though its cleaved forms will present at different molecular weights depending on the specific proteolytic processing.

How can researchers distinguish between cleaved and uncleaved forms of ITGA7 in experimental settings?

Distinguishing between cleaved and uncleaved forms of ITGA7 requires careful experimental design. The Cleaved-ITGA7 (E959) antibody is specifically designed to recognize the neo-epitope exposed after proteolytic cleavage at glutamic acid 959 (E959) . To effectively differentiate between these forms:

• Western Blot Analysis: Run samples alongside appropriate molecular weight markers. The uncleaved full-length ITGA7 appears at approximately 128.9 kDa, while the cleaved forms will show distinct bands corresponding to the heavy chain and the light chain (containing the E959 cleavage site) .

• Validation Controls: Include positive controls such as COS7 cells treated with etoposide (25μM for 1 hour), which has been shown to induce ITGA7 cleavage . Negative controls should include either untreated cells or samples where the antibody has been pre-absorbed with the immunizing peptide.

• Multiple Antibody Approach: Use both the Cleaved-ITGA7 (E959) antibody and antibodies targeting other regions of ITGA7 (such as the extracellular domain antibody clone 9.1) to create a comprehensive profile of ITGA7 status in your samples.

• Densitometric Analysis: Quantify the ratio of cleaved to uncleaved forms using densitometry software to assess the extent of proteolytic processing across different experimental conditions.

What is the significance of ITGA7 cleavage in pathological contexts, and how can Cleaved-ITGA7 (E959) antibody contribute to this research?

ITGA7 cleavage represents an important post-translational modification that may alter integrin signaling and function. While the complete physiological and pathological implications remain under investigation, several research directions can be pursued using the Cleaved-ITGA7 (E959) antibody:

• Cancer Research: Given that alpha 7/beta-1 integrin is expressed in certain tumor cells , the cleaved form may play a role in tumor progression, invasion, or metastasis. The Cleaved-ITGA7 (E959) antibody can help quantify the relative abundance of cleaved forms in tumor versus normal tissue samples.

• Muscle Pathologies: Since ITGA7 is prominently expressed in skeletal and cardiac muscle , alterations in its cleavage pattern may be relevant to muscular dystrophies, cardiomyopathies, or other muscle-related disorders. The antibody allows for specific tracking of the cleaved form in disease models.

• Signaling Pathway Analysis: Cleaved integrins often exhibit altered signaling properties. Researchers can use the antibody in combination with phospho-specific antibodies to downstream signaling molecules to assess how cleavage affects integrin-mediated signaling pathways.

• Drug Development: The antibody can be used to screen compounds that modulate ITGA7 cleavage, potentially identifying therapeutic candidates for conditions where aberrant integrin processing is implicated.

How does etoposide treatment affect ITGA7 cleavage, and what experimental design is optimal for studying this phenomenon?

Based on the Western blot analysis information provided in the datasheet, etoposide treatment (25μM for 1 hour) of COS7 cells induces ITGA7 cleavage . To optimally study this phenomenon:

• Dose-Response Analysis: Treat cells with varying concentrations of etoposide (e.g., 5μM, 15μM, 25μM, 50μM) to determine the dose-dependency of ITGA7 cleavage.

• Time-Course Experiment: Expose cells to 25μM etoposide for different durations (30 minutes, 1 hour, 3 hours, 6 hours) to establish the kinetics of cleavage.

• Mechanism Investigation: Pre-treat cells with various protease inhibitors (caspase inhibitors, matrix metalloproteinase inhibitors, etc.) before etoposide exposure to identify the proteases responsible for cleavage.

• Context Dependence: Compare etoposide-induced ITGA7 cleavage across different cell types that express ITGA7 (muscle cells, various cancer cell lines) to assess tissue-specific variations in this response.

• Functional Consequences: Assess downstream effects of etoposide-induced ITGA7 cleavage on cell adhesion, migration, and survival using appropriate functional assays.

What are the optimal protocols for detecting Cleaved-ITGA7 using Western blot analysis?

For optimal Western blot detection of Cleaved-ITGA7:

• Sample Preparation:

  • Extract total protein using a lysis buffer containing protease inhibitors to prevent artificial cleavage during processing

  • Include phosphatase inhibitors if investigating phosphorylation-dependent cleavage mechanisms

  • Determine protein concentration using Bradford or BCA assay

  • Load 20-50μg of total protein per lane

• Gel Electrophoresis:

  • Use 8-10% SDS-PAGE gels to ensure proper resolution of both cleaved and uncleaved forms

  • Include appropriate molecular weight markers (covering 25-150 kDa range)

  • Run at 100V until samples enter resolving gel, then increase to 150V

• Transfer and Blocking:

  • Transfer to PVDF membrane (preferred over nitrocellulose for detection of cleaved forms)

  • Transfer at 100V for 90 minutes in cold transfer buffer containing 20% methanol

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Antibody Incubation:

  • Dilute Cleaved-ITGA7 (E959) antibody to 1:1000 in 5% BSA in TBST

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3 times with TBST, 5 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit) at 1:5000 dilution for 1 hour at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) reagents

  • Expose to X-ray film or use digital imaging system

  • For validation, strip and reprobe with antibodies against total ITGA7

How can researchers optimize ELISA protocols for detecting Cleaved-ITGA7 (E959)?

To optimize ELISA protocols for Cleaved-ITGA7 detection:

• Plate Coating:

  • Coat 96-well plates with capture antibody (anti-ITGA7 that does not compete with the E959 epitope) at 1-5μg/ml in coating buffer (carbonate-bicarbonate, pH 9.6)

  • Incubate overnight at 4°C

  • Wash 3 times with PBST

• Blocking and Sample Addition:

  • Block with 3% BSA in PBS for 2 hours at room temperature

  • Add samples and standards (serially diluted recombinant ITGA7 fragments)

  • Incubate for 2 hours at room temperature or overnight at 4°C

• Detection Antibody:

  • Dilute Cleaved-ITGA7 (E959) antibody to 1:40000 in 1% BSA in PBST

  • Incubate for 2 hours at room temperature

  • Wash 4-5 times with PBST

• Signal Development:

  • Add appropriately diluted HRP-conjugated secondary antibody

  • Incubate for 1 hour at room temperature

  • Wash 5 times with PBST

  • Add TMB substrate and monitor color development

  • Stop reaction with 2N H₂SO₄ and read absorbance at 450nm

• Validation Controls:

  • Include lysates from etoposide-treated cells as positive controls

  • Use peptide competition (pre-absorbing the antibody with immunizing peptide) as specificity controls

What experimental conditions affect the reproducibility of results when using Cleaved-ITGA7 (E959) antibody?

Several factors can significantly impact result reproducibility:

• Sample Handling:

  • Proteolytic degradation during sample preparation can create artificial cleavage products

  • Always prepare fresh lysates or store properly at -80°C with protease inhibitors

  • Avoid multiple freeze-thaw cycles of protein samples

• Antibody Storage:

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

  • Avoid repeated freeze-thaw cycles

  • Aliquot the antibody upon first thaw for long-term storage

• Batch Effects:

  • Lot-to-lot variability in polyclonal antibodies can affect detection sensitivity

  • Include internal standards across experiments to normalize between different antibody lots

  • Document lot numbers used for each experiment

• Cell Culture Conditions:

  • Serum starvation, cell density, and passage number can all affect integrin expression and processing

  • Standardize cell culture protocols across experiments

  • Document and control for these variables when interpreting results

• Induction Protocols:

  • Variation in etoposide quality, duration of treatment, and cell responsiveness

  • Establish positive controls for each experimental batch

  • Include time-matched vehicle controls

What are common challenges in detecting Cleaved-ITGA7 and their solutions?

When troubleshooting, always include appropriate controls:

• Positive control: Etoposide-treated COS7 cells

• Negative control: Same samples with antibody pre-absorbed with immunizing peptide

• Loading control: Housekeeping protein antibody (β-actin, GAPDH) to ensure equal loading

How can researchers integrate Cleaved-ITGA7 (E959) antibody with other experimental techniques?

The Cleaved-ITGA7 (E959) antibody can be effectively integrated with multiple techniques:

• Immunofluorescence/Immunocytochemistry:

  • Though not explicitly validated in the datasheet, many polyclonal antibodies can work in IF/ICC

  • Start with a 1:200 dilution and optimize as needed

  • Co-stain with markers for cellular compartments to determine subcellular localization of cleaved ITGA7

  • Use confocal microscopy for high-resolution localization studies

• Immunoprecipitation:

  • Use the antibody to pull down cleaved ITGA7 forms

  • Perform co-IP to identify binding partners specific to the cleaved form

  • Follow with mass spectrometry to characterize the complete interactome

• Chromatin Immunoprecipitation (ChIP):

  • If nuclear translocation of cleaved ITGA7 fragments is suspected, ChIP can identify any DNA binding properties

  • Would require careful validation as this is not a standard application for integrin antibodies

• Flow Cytometry:

  • For detecting potential cell surface expression of cleaved forms

  • Requires cell permeabilization protocols if the epitope is intracellular

  • Start with 1:100 dilution and titrate for optimal signal-to-noise ratio

• Mass Spectrometry Integration:

  • Use antibody for enrichment of cleaved forms prior to MS analysis

  • Helps identify exact cleavage sites and potential post-translational modifications

What approaches can resolve data inconsistencies when comparing results from different ITGA7 antibodies?

When different ITGA7 antibodies yield seemingly contradictory results:

• Epitope Mapping:

  • Compare the exact epitopes recognized by each antibody

  • Cleaved-ITGA7 (E959) targets amino acids 910-990 at the C-terminus

  • The 9.1 ITGA7 monoclonal antibody recognizes the extracellular domain

  • These target different regions and may give complementary rather than contradictory information

• Validation with Recombinant Proteins:

  • Test antibodies against recombinant ITGA7 fragments representing different domains

  • Include both cleaved and uncleaved forms in validation experiments

  • Create a reactivity profile for each antibody

• Peptide Competition Assays:

  • Pre-absorb each antibody with its specific immunizing peptide

  • This confirms signal specificity and helps identify non-specific binding

• Knockout/Knockdown Controls:

  • Use ITGA7 knockout or knockdown systems to confirm antibody specificity

  • The disappearance of signal validates antibody specificity

  • Partial signal reduction in knockdowns can help quantify specificity

• Cross-Platform Validation:

  • Compare protein detection across multiple methods (Western blot, ELISA, immunofluorescence)

  • Consistent patterns across methods increase confidence in results

  • Technique-specific discrepancies may reveal methodological limitations

What emerging research areas can benefit from using Cleaved-ITGA7 (E959) antibody?

The Cleaved-ITGA7 (E959) antibody opens opportunities for several cutting-edge research directions:

• Single-Cell Analysis:

  • Investigating heterogeneity in ITGA7 cleavage patterns within apparently homogeneous tissues

  • Combining with single-cell RNA-seq to correlate cleavage events with transcriptional signatures

  • Development of flow cytometry protocols for high-throughput screening of cleaved ITGA7 in cell populations

• Protease Network Mapping:

  • Identifying the specific proteases responsible for E959 cleavage under different physiological and pathological conditions

  • Characterizing how these proteolytic networks are dysregulated in disease states

  • Developing targeted inhibitors of pathological ITGA7 cleavage

• Mechanobiology:

  • Exploring how mechanical forces affect ITGA7 cleavage, particularly in muscle tissues where mechanical stress is prominent

  • Determining if cleaved forms alter mechanotransduction properties of cells

  • Investigating the role of ITGA7 cleavage in muscle adaptation to exercise or disuse

• Extracellular Vesicle Research:

  • Examining if cleaved ITGA7 fragments are packaged into extracellular vesicles

  • Determining if such vesicles serve as intercellular communication signals

  • Evaluating potential as biomarkers in liquid biopsies

• Regenerative Medicine:

  • Assessing the role of ITGA7 cleavage in stem cell maintenance and differentiation

  • Developing strategies to modulate ITGA7 cleavage to enhance tissue regeneration

  • Creating engineered microenvironments with controlled integrin cleavage properties

How might ongoing integrin research influence our understanding of ITGA7 cleavage patterns?

Current trends in integrin research suggest several areas that may enhance our understanding of ITGA7 cleavage:

• Structural Biology Advances:

  • Cryo-EM studies of full-length and cleaved integrin conformations

  • Molecular dynamics simulations of how cleavage affects integrin activation states

  • Structural insights into how cleavage modifies interaction with ECM components and intracellular binding partners

• Integrin Interactome Expansion:

  • Identification of molecules that selectively interact with cleaved versus intact ITGA7

  • Characterizing how these differential interactions affect downstream signaling

  • Mapping the dynamic interactome changes following cleavage events

• Cross-Talk with Other Receptors:

  • Understanding how ITGA7 cleavage influences signaling from growth factor receptors, mechanosensors, and other adhesion molecules

  • Identifying integrative signaling nodes that respond to cleaved integrin fragments

  • Developing mathematical models of receptor cross-talk networks

• Therapeutic Targeting Strategies:

  • Design of conformation-specific inhibitors or activators that discriminate between cleaved and intact forms

  • Development of proteolysis-targeting chimeras (PROTACs) to selectively degrade specific integrin forms

  • Creation of synthetic biology approaches to control integrin cleavage with spatiotemporal precision