HRP conjugation enhances detection sensitivity by linking the enzyme to antibodies via optimized protocols:
Periodate Oxidation: Sodium metaperiodate oxidizes HRP’s carbohydrate moieties, forming reactive aldehydes for antibody binding .
Lyophilization: A modified method involves lyophilizing activated HRP before conjugation, improving stability and antibody-binding capacity. This approach increases assay sensitivity (detection at 1:5,000 dilution vs. 1:25 in classical methods) .
Conjugation Method | Key Advantage | Sensitivity (ELISA) |
---|---|---|
Classical | Simplicity | 1:25 dilution |
Modified (Lyophilized) | Enhanced HRP-antibody binding | 1:5,000 dilution |
Myocardial Injury: KBTBD7 promotes inflammation via p38/NF-κB signaling. Crocin, a natural compound, inhibits KBTBD7, reducing cytokine release (IL-1β, TNFα) and apoptosis in rat models .
Cancer Progression: In NSCLC, KBTBD7 degrades PTEN via ubiquitination, activating EGFR/PI3K/AKT pathways to drive proliferation and metastasis .
Western Blot: Detects KBTBD7 in cell lysates, confirming overexpression in NSCLC tissues .
ELISA: Lyophilized HRP conjugates enable antigen detection as low as 1.5 ng .
While KBTBD7 antibodies enable critical insights into disease mechanisms, challenges include batch variability in polyclonal preparations and the need for broader validation across cancer subtypes . Future studies should explore therapeutic targeting of KBTBD7 in preclinical models.
This KBTBD7 antibody, conjugated with horseradish peroxidase (HRP), targets the KBTBD7 protein. KBTBD7 functions as a substrate adapter within the CUL3(KBTBD6/7) E3 ubiquitin ligase complex. This complex facilitates the ubiquitination and subsequent proteasomal degradation of the RAC1 guanine exchange factor (GEF), TIAM1. Through this regulation of TIAM1 ubiquitination, KBTBD7 controls RAC1 signal transduction and influences downstream processes such as cytoskeletal organization, cell migration, and proliferation. The membrane-associated protein GABARAP is required for TIAM1 ubiquitination and may locally restrict the activity of the CUL3(KBTBD6/7) complex.
Key research findings supporting KBTBD7's function include:
KBTBD7 functions as part of the CUL3(KBTBD6/7) E3 ubiquitin ligase complex, serving as a substrate adapter for the RAC1 guanine exchange factor (GEF) TIAM1. It mediates 'Lys-48' ubiquitination and proteasomal degradation of TIAM1, thereby regulating RAC1 signal transduction . This regulation impacts downstream biological processes including cytoskeleton organization, cell migration, and cell proliferation . Recent research also indicates that KBTBD7 enhances ubiquitin-dependent degradation of PTEN, thus activating EGFR/PI3K/AKT signaling pathways in NSCLC cells .
KBTBD7 antibody, HRP conjugated, can be used in multiple experimental applications including:
Western blot (WB)
Immunohistochemistry on paraffin-embedded tissues (IHC-P)
Immunohistochemistry on frozen sections (IHC-F)
When using the antibody for Western blot applications, a dilution of 1/1000 has been successfully employed with human cell lines such as HL-60 (human promyelocytic leukemia cell line) .
Commercial KBTBD7 antibodies have been confirmed to react with human samples . Some antibodies also demonstrate reactivity with mouse and rat samples . Before application in non-human experimental models, validation experiments should be conducted to confirm cross-reactivity and specificity in the target species.
To maintain optimal activity, KBTBD7 antibody with HRP conjugation should be stored according to manufacturer recommendations. Generally, this involves:
Storage at -20°C for long-term preservation
Avoidance of repeated freeze/thaw cycles that can degrade antibody quality and reduce sensitivity
If working with aliquots, proper storage in single-use volumes to minimize freeze/thaw cycles
Immunohistochemical studies of 104 paired NSCLC and peritumoral normal specimens revealed that KBTBD7 is highly expressed in NSCLC tissues compared to adjacent non-cancerous tissues . This overexpression positively correlates with several clinicopathological characteristics:
Clinicopathological Parameter | Correlation with KBTBD7 Expression | p-value |
---|---|---|
Histological type (squamous cell carcinoma vs. adenocarcinoma) | Positive | 0.028 |
p-TNM stage | Positive | 0.019 |
Lymph node metastasis | Positive | 0.034 |
Tumor size (>3cm vs. ≤3cm) | Positive | <0.01 |
These findings suggest KBTBD7 may serve as a potential biomarker for NSCLC progression and could represent a therapeutic target .
When investigating KBTBD7's role in ubiquitin-dependent protein degradation (such as PTEN), researchers should consider the following methodological approach:
Establish stable KBTBD7 knockdown cell lines (e.g., in NSCLC cell lines like A549 or H1299)
Transfect cells with Ub-HA plasmid to enable detection of ubiquitination
Pre-treat cells with proteasome inhibitor MG-132 (typically 12 hours before collection) to prevent degradation of ubiquitinated proteins
Perform immunoprecipitation using antibodies against the target protein (e.g., anti-PTEN)
Evaluate ubiquitination levels using anti-HA immunoblotting via Western blot analysis
This approach allows for assessment of how KBTBD7 affects the ubiquitination and subsequent degradation of target proteins.
To determine whether KBTBD7 regulates its target proteins at the transcriptional or post-translational level, researchers should implement a dual analysis approach:
Transcriptional analysis: Perform RT-PCR to quantify mRNA levels of target proteins (e.g., PTEN) in both KBTBD7-suppressed and control cells. For example, studies show no change in PTEN mRNA levels when KBTBD7 is suppressed, suggesting regulation occurs post-transcriptionally .
Protein interaction analysis: Conduct co-immunoprecipitation assays to verify direct protein-protein interactions between KBTBD7 and suspected targets .
Protein degradation analysis: Use proteasome inhibitors (e.g., MG132) along with cycloheximide chase assays to assess protein stability and degradation rates.
Ubiquitination assays: As detailed in question 2.2, these assays can confirm KBTBD7's role in ubiquitin-mediated protein degradation.
The combination of these techniques provides comprehensive evidence of KBTBD7's regulatory mechanisms.
For optimal immunohistochemical detection of KBTBD7 in tissue specimens, researchers should consider the following protocol:
Sample preparation:
Use tissue specimens sliced at approximately 4 μm thickness
Heat specimens at 70°C for 4–6 h before immunohistochemical staining
Antigen retrieval:
Perform xylene deparaffinization followed by gradient alcohol hydration
Conduct heat-induced epitope retrieval using slightly boiling EDTA repair solution for 20 minutes
Antibody incubation:
Use anti-KBTBD7 antibody at an optimized dilution (e.g., 1:100)
Incubate according to the manufacturer's recommended duration and temperature
Scoring system implementation:
Assess staining intensity on a scale of 0-3: 0 (no staining), 1 (weak, light yellow), 2 (medium, yellow), 3 (strong, dark yellow/brown)
Evaluate the stained area on a scale of 1-4: 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), 4 (76%–100%)
Calculate final score by multiplying intensity and area scores (range: 0-12)
Consider specimens with scores >6 as KBTBD7-positive and those with scores ≤6 as KBTBD7-negative
When performing Western blot experiments with KBTBD7 antibody, HRP conjugated, the following controls should be included:
Positive control: Include cell lines known to express KBTBD7, such as SK, A549, H1975, H1299, or HCC827 NSCLC cell lines, which demonstrate high expression levels compared to normal bronchial epithelial cells (HBE) .
Negative control: Include cell lines with low or no KBTBD7 expression, or use siRNA/shRNA knockdown samples as negative controls.
Loading control: Include detection of housekeeping proteins such as GAPDH to ensure equal loading across lanes.
Antibody specificity control: Consider running a competition assay with the immunizing peptide to confirm specificity.
Molecular weight marker: Include to confirm that the detected band corresponds to the expected molecular weight of KBTBD7.
For RT-PCR analysis of KBTBD7 expression, the following primer sequences have been successfully utilized:
KBTBD7:
Forward: 5′-AGACGCCTTCGACCATCAC-3′
Reverse: 5′-GAATTGAACCCATTCGGCTGA-3′
For comparison with related genes or targets, these additional primers may be useful:
PTEN:
Forward: 5′-TGGATTCGACTTAGACTTGACCT-3′
Reverse: 5′-GGTGGGTTATGGTCTTCAAAAGG-3′
EGFR:
Forward: 5′-GGAGAACTGCCAGAAACTGACC-3′
Reverse: 5′-GCCTACAGCACACTGGTTG-3′
GAPDH (reference gene):
KBTBD7 antibodies can be employed to investigate the relationship between KBTBD7 expression and cancer cell behavior through multiple experimental approaches:
Expression analysis in clinical samples: Use immunohistochemistry with KBTBD7 antibody to correlate expression levels with tumor characteristics and patient outcomes .
Functional studies in cell lines:
Create KBTBD7 knockdown cell lines and examine effects on proliferation (using CCK-8 and colony formation assays)
Assess invasion capabilities using Transwell assays
Examine expression of proliferation-related proteins (CCNE1, CDK4, P27) and invasion-related proteins (MMP-9, Claudin-1, Rock1, ZEB-1, E-cadherin) by Western blot following KBTBD7 suppression
Signaling pathway analysis: Investigate how KBTBD7 affects key oncogenic signaling pathways, such as EGFR/PI3K/AKT, by examining phosphorylation status of pathway components after KBTBD7 modulation .
When conducting immunofluorescence studies to determine KBTBD7's subcellular localization:
Cell preparation: Both NSCLC cell lines and normal control cells (e.g., HBE) should be included for comparative analysis.
Antibody selection: Use primary antibodies specific to KBTBD7 followed by fluorophore-conjugated secondary antibodies if the primary antibody is not directly conjugated.
Co-localization markers: Include markers for specific cellular compartments (nuclear, cytoplasmic, membrane) to precisely determine localization.
Microscopy parameters: Use confocal microscopy for higher resolution imaging to accurately determine subcellular distribution.
Analysis and interpretation: Based on existing research, expect to observe predominantly cytoplasmic localization of KBTBD7 in both NSCLC and HBE cell lines .
To verify KBTBD7's function in the ubiquitin-proteasome pathway:
Protein-protein interaction studies:
Ubiquitination assays:
Protein stability analysis:
Perform cycloheximide chase assays in cells with normal or altered KBTBD7 expression
Monitor degradation rates of target proteins over time
E3 ligase activity reconstitution:
Conduct in vitro ubiquitination assays with purified components including KBTBD7, CUL3, ubiquitin, E1, E2, and substrate proteins
This comprehensive approach provides strong evidence for KBTBD7's role as a component of the E3 ubiquitin ligase complex.
When working with KBTBD7 antibody (HRP conjugated) in Western blotting, researchers may encounter the following challenges:
High background signal:
Weak or no signal:
Multiple bands or unexpected band sizes:
Verify antibody specificity with manufacturer
Consider post-translational modifications or splice variants
Include appropriate positive controls with known KBTBD7 expression
Inconsistent results:
When investigating KBTBD7's interaction with PTEN and its implications in cancer:
Cell line selection:
Use multiple NSCLC cell lines (e.g., A549, H1299) to ensure robustness of findings
Include cell lines with varying baseline PTEN expression levels
KBTBD7 modulation approaches:
Employ both knockdown (siRNA, shRNA) and overexpression systems
Consider inducible systems for temporal control of expression
Interaction validation:
Perform reciprocal co-immunoprecipitation (IP KBTBD7, blot PTEN and vice versa)
Use proximity ligation assays for in situ confirmation
Consider GST pull-down assays with recombinant proteins to confirm direct interaction
Functional consequences:
Monitor PTEN protein levels and phosphorylation status of downstream targets (AKT)
Assess ubiquitination status of PTEN following proteasome inhibition
Examine phenotypic outcomes (proliferation, invasion) following PTEN rescue in KBTBD7-overexpressing cells
Domain mapping:
Generate truncation or point mutation constructs to identify critical interaction domains
Assess how mutations affect KBTBD7-mediated PTEN degradation
These considerations ensure comprehensive characterization of the KBTBD7-PTEN regulatory axis in cancer contexts.