Cleaved-ITGA6 (E942) Antibody

What applications is the Cleaved-ITGA6 (E942) antibody validated for?

The Cleaved-ITGA6 (E942) antibody has been validated primarily for Western Blot (WB) and ELISA applications . In Western blot applications, the recommended dilution range is 1:500-1:2000, while for ELISA, the recommended dilution is 1:10000 . The antibody has demonstrated reactivity with human, rat, and mouse samples, making it versatile for comparative studies across these species . It has not yet been extensively validated for other applications such as immunohistochemistry, immunofluorescence, or flow cytometry based on the available product information.

What are the optimal storage conditions for Cleaved-ITGA6 (E942) antibody?

For optimal longevity and performance of the Cleaved-ITGA6 (E942) antibody, researchers should store it at -20°C for up to 1 year from the date of receipt . The antibody is typically provided in a formulation containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide as a preservative . It is crucial to avoid repeated freeze-thaw cycles as this can lead to antibody degradation and loss of activity . For laboratories that require frequent use of the antibody, it is recommended to prepare small working aliquots and keep only the working aliquot at 4°C while maintaining the stock at -20°C.

How can researchers validate the specificity of Cleaved-ITGA6 (E942) antibody?

To validate the specificity of the Cleaved-ITGA6 (E942) antibody, researchers should implement several control experiments:

• Peptide competition assay: Use the synthesized immunogenic peptide (amino acids 923-972 of human ITGA6) to block antibody binding. As shown in product documentation, "The lane on the right is blocked with the synthesized peptide" demonstrates reduced signal with peptide competition .

• Positive control samples: Use cells treated with agents known to induce ITGA6 cleavage, such as etoposide (25μM for 24h in HeLa cells) .

• Molecular weight verification: The cleaved form of ITGA6 (α6p) has a molecular weight of approximately 70 kDa, distinct from the full-length protein (120-150 kDa) .

• siRNA knockdown: Targeted knockdown of ITGA6 using specific siRNA should reduce the signal detected by the antibody.

• Multiple cell line comparison: Test the antibody across cell lines with known differential expression of ITGA6 to verify consistent detection patterns.

What are the key considerations for sample preparation when using Cleaved-ITGA6 (E942) antibody?

When preparing samples for analysis with Cleaved-ITGA6 (E942) antibody, researchers should consider:

• Preservation of post-translational modifications: Use protease inhibitors and phosphatase inhibitors in lysis buffers to preserve the native state of the protein.

• Denaturation conditions: For Western blot applications, standard SDS-PAGE conditions with reducing agents are appropriate as the antibody was generated against a linear epitope.

• Sample loading: Ensure equal loading (20-50 μg of total protein per lane) to enable accurate quantification.

• Positive controls: Include samples from cells treated with stimuli known to induce ITGA6 cleavage, such as etoposide treatment in HeLa cells .

• Membrane selection: PVDF membranes are generally recommended for optimal protein transfer and antibody binding.

• Blocking optimization: 5% non-fat dry milk or BSA in TBST is typically suitable for blocking non-specific binding sites.

How can researchers distinguish between full-length ITGA6 and the cleaved form (α6p) in complex biological samples?

Distinguishing between full-length ITGA6 and its cleaved form requires careful experimental design:

• Antibody selection: Use both N-terminal and C-terminal domain-specific antibodies in parallel. Full-length ITGA6 will be detected by both antibodies, while α6p will only be detected by C-terminal antibodies like Cleaved-ITGA6 (E942).

• Molecular weight discrimination: Full-length ITGA6 appears at 150-125 kDa on Western blots, while α6p appears at approximately 70 kDa .

• Two-dimensional gel electrophoresis: This approach can help distinguish the cleaved form based on both molecular weight and isoelectric point.

• Mass spectrometry: Peptide mapping using mass spectrometry can definitively identify the cleavage site and distinguish between full-length and cleaved forms.

• Sequential immunoprecipitation: Use an N-terminal antibody to deplete full-length ITGA6, then probe the supernatant with Cleaved-ITGA6 (E942) antibody to detect only cleaved forms.

The following table summarizes the distinguishing features:

What are the implications of ITGA6 cleavage at E942 in cancer progression and metastasis?

The cleavage of ITGA6 at E942 has several significant implications for cancer biology:

Research has shown that the uPA-mediated proteolytic removal of the laminin-binding domain creates α6p, which lacks the extracellular ligand-binding domain but retains signaling capabilities . The ablation of ITGA6 from Ph+ acute lymphocytic leukemia cells results in cell cycle suppression and an increase in the proportion of cells in the G0/G1 and G2/M phases . Long-term loss of ITGA6 has been related to caspase activation and increment in apoptosis over time, associated with an increase in cleaved poly (ADP-ribose) polymerase and p53 expression .

How does phosphorylation affect ITGA6 susceptibility to cleavage at E942?

Phosphorylation plays a critical role in regulating ITGA6 function and susceptibility to cleavage:

• Post-translational regulation: ITGA6 contains phosphorylation sites that can influence its conformation and interaction with other proteins. Specifically, isoforms containing segment A are the major targets for PMA-induced phosphorylation at 'Ser-1103' of isoform alpha-6X1X2A .

• Kinase involvement: Research indicates that phosphorylation by specific kinases can affect ITGA6 stability and processing. For example, after the ablation of ITGA6 in BCR-ABL (Ph+) acute lymphocytic leukemia cells, the phosphorylation of the docking protein CASL increases, while the phosphorylation of SFK substrates decreases .

• Functional consequences: While phosphorylation is not required for the induction of integrin alpha-6A/beta-1 high affinity, it may reduce the affinity for ligand . This altered affinity could expose or mask protease recognition sites.

• Cross-talk with other signaling pathways: In pancreatic and melanoma cancers, active KRas and BRAF stimulate the production of pro-tumorigenic ITGA6 through the ERK pathway , suggesting that phosphorylation events downstream of these oncogenes may influence ITGA6 processing.

• Therapeutic implications: Understanding the phosphorylation-cleavage relationship could reveal intervention points. For instance, Src kinase inhibitors diminish the production of ITGA6 in B-acute lymphocytic leukemia cells .

What methodological approaches can be used to study the dynamic regulation of ITGA6 cleavage in live cells?

Several advanced methodological approaches can be employed to study dynamic ITGA6 cleavage:

• FRET-based biosensors: Design fluorescence resonance energy transfer (FRET) constructs with fluorophores flanking the E942 cleavage site to monitor cleavage events in real-time.

• Split-GFP complementation assays: Engineer ITGA6 with split-GFP fragments positioned such that cleavage alters complementation efficiency, providing a visual readout of cleavage status.

• Live-cell immunostaining: Use non-permeabilizing conditions with fluorescently labeled antibodies against extracellular ITGA6 epitopes to track surface expression dynamics.

• Photoactivatable or photoconvertible fluorescent protein fusions: Tag ITGA6 with these specialized fluorescent proteins to pulse-chase specific populations and track their processing over time.

• CRISPR-Cas9 knock-in of endogenous tags: Insert small epitope tags or fluorescent proteins at the endogenous ITGA6 locus to track native protein without overexpression artifacts.

• Bimolecular fluorescence complementation (BiFC): Use to visualize interactions between ITGA6 and potential binding partners that may influence cleavage susceptibility.

• Fluorescence recovery after photobleaching (FRAP): Apply to study how ITGA6 mobility in the membrane might correlate with cleavage events.

• Single-molecule tracking: Implement to follow individual ITGA6 molecules and identify spatial restrictions or patterns associated with cleavage events.

How can the Cleaved-ITGA6 (E942) antibody be employed in multiplex immunoassays to study integrin signaling networks?

The Cleaved-ITGA6 (E942) antibody can be strategically incorporated into multiplex assays:

• Multiplex Western blotting: Use fluorescently labeled secondary antibodies with different spectra to simultaneously detect cleaved ITGA6 alongside binding partners or downstream signaling molecules. This approach can reveal correlations between ITGA6 cleavage and activation of specific signaling pathways.

• Proximity ligation assay (PLA): Combine Cleaved-ITGA6 (E942) antibody with antibodies against potential interaction partners to visualize and quantify protein interactions within 40 nm proximity in fixed cells.

• Mass cytometry (CyTOF): Label Cleaved-ITGA6 (E942) antibody with rare earth metals for high-dimensional analysis of integrin signaling in heterogeneous cell populations without spectral overlap limitations.

• Reverse phase protein array (RPPA): Apply to quantitatively assess cleaved ITGA6 levels across large sample sets in parallel with dozens of signaling proteins.

• Sequential immunoprecipitation workflows: Use Cleaved-ITGA6 (E942) antibody to isolate protein complexes containing the cleaved form, followed by mass spectrometry to identify associated proteins.

• Multiplexed immunofluorescence: Implement for tissue sections to spatially resolve cleaved ITGA6 in relation to other markers of interest, including β1 or β4 integrin subunits that associate with ITGA6 to form functional heterodimers .

• Single-cell Western blot: Apply to correlate cleaved ITGA6 levels with other signaling proteins at the single-cell level, revealing population heterogeneity.

• Bead-based multiplex assays: Develop for simultaneous quantification of cleaved ITGA6 and related signaling proteins in solution-phase samples.

What are the optimal experimental designs for investigating the relationship between ITGA6 cleavage and cancer stem cell properties?

When designing experiments to investigate the relationship between ITGA6 cleavage and cancer stem cell properties, researchers should consider:

• Cell model selection: Use established cancer stem cell models with well-characterized ITGA6 expression. The search results indicate that ITGA6 (CD49f) is a documented cancer stem cell marker .

• Cleavage modulation approaches:

  • Genetic: Express cleavage-resistant ITGA6 mutants (mutation at E942)

  • Pharmacological: Use uPA inhibitors to prevent cleavage

  • siRNA/shRNA: Target uPA or other proteases involved in cleavage

• Stemness assays:

  • Sphere formation capability in low-attachment conditions

  • Expression of stemness markers (OCT4, SOX2, NANOG)

  • Limiting dilution assays for tumor initiation potential

  • Side population analysis for drug efflux capacity

  • Serial transplantation studies in appropriate animal models

• Signaling pathway analysis:

  • Monitor key pathways known to regulate stemness (Wnt, Notch, Hedgehog)

  • Investigate the effect of ITGA6 cleavage on these pathways using reporter assays

  • Assess how cleavage affects interaction with key signaling partners

• Isoform-specific considerations: Distinguish between ITGA6-A and ITGA6-B splice variants, as their roles in stemness differ. Human embryonic stem cells express both spliced isoforms, but ITGA6-A is required to inhibit FAK activity and prevent differentiation, while ITGA6-B is responsible for maintaining stemness in breast cancer stem cells .

• Therapeutic resistance correlations: Examine whether ITGA6 cleavage status correlates with resistance to conventional therapies, as integrin signaling dysregulation can be involved in resistance to therapeutic interventions .

• In vivo validation: Confirm in vitro findings through xenograft studies comparing tumor-initiating capacity and growth dynamics of cells expressing wild-type versus cleavage-resistant ITGA6.

What considerations should be made when comparing results obtained with Cleaved-ITGA6 (E942) antibody across different cell types or tissues?

When comparing results obtained with Cleaved-ITGA6 (E942) antibody across different experimental systems, researchers should consider:

• Baseline expression levels: Different cell types and tissues have varying baseline expression of ITGA6. According to search results, the expression levels of ITGA6 by thymus epithelial cells are relevant in the general control of MHC expression .

• Isoform distribution: The distribution of ITGA6 splice variants (ITGA6-A and ITGA6-B) varies between tissues and can affect antibody recognition and biological function .

• Heterodimer partners: ITGA6 can associate with either β1 or β4 subunits to form integrins that interact with extracellular matrix proteins including members of the laminin family . The distribution of these heterodimer partners may vary between cell types.

• Proteolytic environment: The presence and activity of proteases responsible for ITGA6 cleavage, particularly uPA, may differ between tissues and affect the relative abundance of cleaved forms.

• Post-translational modifications: Variations in phosphorylation and other modifications can affect antibody binding and detection sensitivity .

• Matrix composition: The extracellular matrix composition varies between tissues and can influence integrin activation and processing.

• Loading controls: Select appropriate loading controls specific to each cell type or tissue being compared.

• Signal normalization strategies: Consider both total ITGA6 and cleaved ITGA6 levels when making comparisons between systems with different baseline expression.

• Validation with alternative methods: Confirm antibody-based findings with orthogonal approaches such as mass spectrometry or functional assays.

• Biological context interpretation: The biological significance of ITGA6 cleavage may vary; for example, in cancer cells it may promote tumorigenesis, while in intestinal epithelial cells, ITGA6 has been categorized as a tumor suppressor because its depletion causes persistent inflammation leading to tumor growth .