ZBTB40 Antibody
Description
Introduction to ZBTB40 Protein and Its Antibodies
ZBTB40 belongs to the ZBTB family of transcriptional regulators that are evolutionarily conserved . These proteins feature several C-terminal C2H2/Krüppel-type zinc finger domains and an N-terminal BTB (broad-complex, tram-track, and bric-a-brac) domain . The zinc finger domains primarily mediate DNA binding, while the BTB domain facilitates protein-protein interactions . ZBTB40 is primarily localized in the nucleus and may be involved in transcriptional regulation .
ZBTB40 antibodies are specialized laboratory reagents developed to detect and analyze the ZBTB40 protein in various research applications. These antibodies bind specifically to ZBTB40, enabling scientists to investigate its expression patterns, subcellular localization, and functional significance in diverse biological processes.
Host and Clonality
The majority of commercial ZBTB40 antibodies are rabbit polyclonal antibodies . Polyclonal antibodies offer the advantage of recognizing multiple epitopes on the ZBTB40 protein, increasing detection sensitivity across various experimental conditions.
Target Regions and Specificity
Commercial ZBTB40 antibodies target different regions of the ZBTB40 protein:
This diversity allows researchers to select antibodies that bind to different functional domains of ZBTB40, depending on their experimental requirements.
Purification Methods
Most ZBTB40 antibodies undergo affinity purification from rabbit antiserum using epitope-specific immunogens . This purification method enhances the specificity of the antibodies by removing non-specific antibodies from the serum.
Applications of ZBTB40 Antibodies
ZBTB40 antibodies serve as versatile tools in multiple research applications, as detailed in the following table:
Western Blotting
Western blotting represents the most common application for ZBTB40 antibodies. The observed molecular weight of ZBTB40 is approximately 138 kDa . ZBTB40 antibodies have successfully detected endogenous levels of ZBTB40 protein in various cell lines including A431, HepG2, L02, and Raji cells .
Immunofluorescence Studies
Immunofluorescence applications have revealed that ZBTB40 is primarily localized in the nucleus, consistent with its putative role in transcriptional regulation . Some antibodies have been validated for immunofluorescence at concentrations ranging from 5-20 μg/mL .
Research Findings on ZBTB40 Function
Recent studies employing ZBTB40 antibodies have significantly advanced our understanding of this protein's biological functions:
ZBTB40 in Telomere Protection
Groundbreaking research has identified ZBTB40 as a telomere-associated protein (TAP) that plays a crucial role in protecting telomeres in human cells, particularly those utilizing the alternative lengthening of telomeres (ALT) mechanism .
ZBTB40 binds to telomeric double-stranded DNA through its N-terminal BTB domain in ALT cells . This binding is fundamental for telomere protection, as demonstrated by functional studies where knockdown or knockout of ZBTB40 resulted in telomere dysfunction-induced foci (TIF) and increased apoptosis .
Further investigations have revealed that ZBTB40 is associated with ALT-associated promyelocytic leukemia nuclear bodies (APBs), which are considered telomere recombination centers . The loss of ZBTB40 induces the accumulation of APBs in U2OS cells, suggesting that ZBTB40 may regulate telomere recombination and lengthening in ALT cells .
While most research on ZBTB40's telomeric function has concentrated on ALT cells, evidence suggests that ZBTB40 may also regulate telomeres in non-ALT cells. Chromatin immunoprecipitation (ChIP)-Seq data indicate an association between ZBTB40 and telomeres in K562 cells, and ZBTB40/telomere colocalization foci have been observed in HeLa cells, both of which are telomerase-positive cell lines .
ZBTB40 in Bone Development and Mineralization
ZBTB40 has been identified as a regulator of osteoblast function and bone mineral density (BMD) . Research has demonstrated that knockdown of ZBTB40 in osteoblasts dramatically reduces mineralization .
In studies using MC3T3-E1 Subclone 4 pre-osteoblast cells, ZBTB40 siRNA-treated cells exhibited a complete absence of mineralized nodule formation, as measured by Alizarin Red staining . Additionally, there was a reduction in alkaline phosphatase (ALP) staining intensity and activity, indicating impaired osteoblast differentiation .
At the molecular level, ZBTB40 siRNA knockdown led to reduced mRNA expression of early osteoblast markers and transcription factors, including Msx2, Col1a1, Runx2, and Sp7 . Upon maturation, there was also a reduction in expression of Bglap, the gene that encodes for the mature osteoblast marker osteocalcin .
To further confirm ZBTB40's role in bone development, researchers created a mouse model using CRISPR/Cas9 technology, generating a strain homozygous for a mutant form of the ZBTB40 protein lacking the BTB domain . Calvarial osteoblasts from these mice showed reduced ALP staining and activity, as well as reduced Alizarin Red staining compared to osteoblasts from wild-type mice, consistent with the findings from siRNA knockdown studies .
ZBTB40 in Spermatogenesis and Male Fertility
Recent studies have implicated ZBTB40 in spermatogenesis and male fertility . ZBTB40 is specifically expressed in mouse spermatocytes, where it colocalizes with γH2AX, a hallmark of chromatin remodeling and inactivation of sex chromosome events during spermatogenesis .
ZBTB40-deficient mice exhibit significantly lower cauda epididymis weight, cauda epididymis/body weight ratio, and testis/body weight ratio compared to wild-type mice . The number of spermatozoa in the cauda epididymis from ZBTB40-deficient mice is remarkably reduced, and more than 95% of these spermatozoa are immotile .
At the cellular level, ZBTB40 deficiency leads to increased apoptosis and inhibited proliferation of male germ cells . TUNEL assays show a significant increase in apoptotic male germ cells in ZBTB40-deficient testes, while the percentage of Ki67-positive germ cells is significantly reduced .
ZBTB40 deficiency also affects sperm morphology, with abnormalities observed in the principal piece of the sperm tail and in acrosome biogenesis . These findings suggest that ZBTB40 plays a critical role in spermatogenesis, and its deficiency leads to morphological and phenotypic abnormalities of spermatocytes and spermatids, resulting in male infertility .
Further supporting ZBTB40's role in male fertility, variants in the ZBTB40 gene have been identified in patients with non-obstructive azoospermia (NOA), a condition characterized by the absence of sperm in the ejaculate due to impaired spermatogenesis .
Antibodies-Online (ABIN3187523)
This rabbit polyclonal antibody targets the C-terminal region of human ZBTB40 and detects endogenous levels of ZBTB40 protein . It is suitable for Western blotting, ELISA, and immunohistochemistry applications . The antibody has been affinity-purified from rabbit antiserum using an epitope-specific immunogen derived from the C-terminal region of human ZBTB40 .
Abcepta (ASC11498)
The ASC11498 antibody can be used for Western blot detection of ZBTB40 at 0.5 μg/mL and for immunofluorescence starting at 5-20 μg/mL . It is human-specific and can recognize at least two known isoforms of ZBTB40 . Importantly, this antibody is not predicted to cross-react with other ZBTB protein family members, ensuring specificity in experimental applications .
St John's Labs (STJ96300)
This antibody targets amino acids 1121-1170 of human ZBTB40 and is reactive against both human and mouse ZBTB40 . It can be used in multiple applications including Western blot (1:500-1:2000), immunohistochemistry (1:100-1:300), immunofluorescence (1:50-200), and ELISA (1:10000) . The antibody is formulated as a liquid in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide .
Proteintech (20026-1-AP)
The 20026-1-AP antibody from Proteintech targets ZBTB40 and shows reactivity with human samples in Western blot and ELISA applications . The observed molecular weight of ZBTB40 with this antibody is 138 kDa . It has been validated in multiple human cell lines including A431, HepG2, and L02 cells . This antibody can recognize all known isoforms of ZBTB40 .
Product Specs
Target Background
- Opposite associations of osteoprotegerin and ZBTB40 polymorphisms with bone mineral density of the hip in postmenopausal Taiwanese women. PMID: 22824048
- Clinical trial of gene-disease association and gene-environment interaction. (HuGE Navigator) PMID: 20379614
- Observational study of gene-disease association. (HuGE Navigator) PMID: 18445777
Q&A
What is ZBTB40 and what are its known biological functions?
ZBTB40 is a transcriptional regulator with multiple emerging biological roles. Research has demonstrated that ZBTB40:
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Functions as a telomere-associated protein (TAP) that binds to telomeric dsDNA through its N-terminal BTB/POZ domain, particularly in ALT (Alternative Lengthening of Telomeres) cells
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Plays a critical role in male fertility and spermatogenesis, with knockout mice exhibiting infertility
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Is expressed specifically in mouse spermatocytes and co-localizes with γH2AX, a hallmark of chromatin remodeling during spermatogenesis
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Contributes to osteoblast activity and bone mineralization, with knockdown leading to reduced mineralization in vitro
Most commercially available ZBTB40 antibodies are rabbit polyclonal antibodies raised against various regions of the protein:
It's worth noting that most vendor-supplied antibodies have been affinity-purified from rabbit antiserum using epitope-specific immunogens, which helps improve specificity .
How can researchers validate ZBTB40 antibody specificity for their applications?
Validation should include multiple complementary approaches:
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Genetic knockdown controls: Compare detection between wild-type samples and those with ZBTB40 knockdown/knockout. Published studies have used ZBTB40 siRNA in U2OS cells to demonstrate specificity .
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Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application. A specific signal should be significantly reduced or eliminated .
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Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of ZBTB40 to confirm results.
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Co-localization studies: For suspected functions (e.g., telomere association), perform dual labeling with established markers. For example, co-localization with telomeric peptide nucleic acid probes or TRF2 antibodies has been used to validate ZBTB40's telomeric association .
What are the challenges in detecting ZBTB40 in different experimental systems?
Several experimental considerations should be addressed:
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Molecular weight variations: While the predicted molecular weight is 138 kDa, expression of different isoforms may result in size variations .
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Cell type-specific expression: ZBTB40 shows cell type-specific localization patterns, particularly in:
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Detection in co-localization studies: When studying telomere association, approximately 30% of U2OS cell nuclei had 5-8 co-localization foci, while only about 5% had more than ten foci , suggesting careful quantification is necessary.
What controls are essential when studying ZBTB40's role in spermatogenesis?
Based on published research methodologies:
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Tissue-specific controls: Compare ZBTB40 staining patterns between testis sections and other tissues where expression is expected to be low or absent .
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Developmental stage comparisons: Evaluate ZBTB40 expression across different stages of spermatogenesis, as it appears specifically in spermatocytes .
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Co-localization with stage-specific markers: Use γH2AX co-staining to identify the association with chromatin remodeling and sex chromosome inactivation events .
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Functional validation: Confirmed phenotypes in ZBTB40 knockout/knockdown models include:
How should researchers approach studying ZBTB40's role in telomere biology?
Based on published methodologies:
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Telomere co-localization assays: Combine ZBTB40 immunofluorescence with telomeric FISH or TRF2 antibody staining to visualize association with telomeres .
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ALT vs. non-ALT cell comparisons: ZBTB40/telomere co-localization is more pronounced in ALT-positive cells (e.g., U2OS, ZOS) compared to non-ALT cells (e.g., HeLa) .
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Functional assessments: Following ZBTB40 knockdown/knockout:
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Measure telomere dysfunction-induced foci (TIF)
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Assess changes in APB (ALT-associated PML bodies)
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Evaluate telomere length alterations
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Research has shown that ZBTB40 knockout in U2OS cells leads to increased TIF formation and APB accumulation, suggesting its role in telomere protection and recombination regulation .
What methodological approaches are recommended for studying ZBTB40's function in bone biology?
Research has established protocols for investigating ZBTB40's role in osteoblast activity:
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In vitro mineralization assays: Compare mineralization capacity (using Alizarin Red staining) between control and ZBTB40-knockdown/knockout osteoblasts .
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Osteoblast differentiation markers: Assess expression of osteoblast-specific genes (Col1a1, Runx2, Sp7) following ZBTB40 manipulation .
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CRISPR/Cas9 models: Generate mouse models with targeted mutations in the BTB domain to study domain-specific functions, as demonstrated with the Zbtb40mut/mut model that lacks the protein-protein interaction domain .
What are the methodological considerations for investigating ZBTB40's role in male fertility?
Based on research findings, several approaches have proven effective:
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Reproductive phenotyping: Assess:
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Cellular analyses:
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Ultrastructural studies: Use transmission electron microscopy (TEM) to examine acrosome biogenesis and other subcellular structures affected by ZBTB40 deficiency .
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Genetic analyses: Screen for ZBTB40 variants in patients with reproductive disorders, particularly non-obstructive azoospermia (NOA), as mutations have been identified in approximately 0.70% of NOA patients .
How should researchers interpret conflicting ZBTB40 localization patterns across different studies?
Discrepancies in localization patterns may arise from:
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Cell-type specificity: ZBTB40 shows distinct localization in:
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Experimental conditions: Different fixation methods, antibody clones, or detection systems may influence subcellular localization patterns.
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Functional states: ZBTB40's localization may be dynamic and influenced by:
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Cell cycle phase
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DNA damage states
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Differentiation stage
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Researchers should carefully document cell types, experimental conditions, and functional states when reporting localization patterns.
What are the implications of ZBTB40 mutations found in clinical samples?
Research has identified several ZBTB40 variants in non-obstructive azoospermia patients:
| Variant Type | Specific Mutation | Predicted Impact | Detection Method |
|---|---|---|---|
| Splicing | c.1025-8T>- (homozygous) | Potentially damaging to protein function | Whole exome sequencing |
| Splicing | c.2833+4T>C (heterozygous) | Potentially damaging to protein function | Whole exome sequencing |
| Missense | c.25C>G (p.Q9E) | Predicted damaging by multiple algorithms | Whole exome sequencing |
| Missense | c.C3257T (p.T1086M) | Predicted damaging by multiple algorithms | Whole exome sequencing |
| Missense | c.G3350A (p.G1117E) | Predicted damaging by multiple algorithms | Whole exome sequencing |
These findings suggest that ZBTB40 mutations may contribute to male infertility, with a frequency of approximately 0.70% (6/856) in NOA patients . Researchers investigating fertility disorders should consider including ZBTB40 in genetic screening panels.
What are promising areas for future ZBTB40 research that would benefit from antibody-based approaches?
Several emerging research directions could benefit from antibody-based methodologies:
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Protein-protein interaction studies: Immunoprecipitation coupled with mass spectrometry to identify ZBTB40 binding partners in different cell types.
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Chromatin immunoprecipitation sequencing (ChIP-seq): To identify genomic binding sites beyond telomeres, particularly in reproductive and bone tissues.
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Single-cell approaches: Examining ZBTB40 expression and localization at the single-cell level during spermatogenesis or osteoblast differentiation.
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Therapeutic applications: Investigating ZBTB40 as a potential target for bone disorders, given its role in mineralized nodule formation and potential relevance to osteoporosis treatment .
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Structural studies: Using antibodies to isolate native ZBTB40 for structural analyses of its BTB domain and zinc finger regions, which are critical for its function .
What methodological advances might improve ZBTB40 research?
Future technical developments could enhance ZBTB40 research:
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Monoclonal antibodies: Development of highly specific monoclonal antibodies against different domains of ZBTB40 would improve consistency across studies.
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Domain-specific antibodies: Antibodies targeting specific functional domains (BTB domain, zinc fingers) could help dissect domain-specific functions.
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Phospho-specific antibodies: As regulatory proteins are often controlled by phosphorylation, antibodies recognizing phosphorylated forms could reveal activation states.
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Live-cell imaging tools: Antibody-based fluorescent probes for tracking ZBTB40 dynamics in living cells could reveal temporal patterns of localization and function.
