PAX6 Recombinant Monoclonal Antibody
Description
Antibody Production and Specificity
Recombinant monoclonal antibodies are generated by cloning DNA sequences from immunoreactive rabbits into plasmid vectors, followed by expression in host systems (e.g., E. coli, mammalian cells). This method ensures lot-to-lot consistency and avoids variability inherent in hybridoma-based production .
Table 2: Western Blot Band Patterns
| Sample | Band Size (kDa) | Antibody | Source |
|---|---|---|---|
| A549 (WT) | 50 | ab109233 (Abcam) | |
| Y79 (Retinoblastoma) | 47 | ab195045 (Abcam) | |
| Mouse Brain | 47 | 67529-1-Ig (PT) |
Immunohistochemistry
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Staining Patterns:
Transcriptional Regulation
PAX6 acts as both an activator and repressor:
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Activation: Binds to promoters of genes like glucagon and somatostatin in pancreatic islets .
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Repression: Competes with PAX4 for promoter binding and inhibits AR-mediated transactivation .
Post-Translational Modifications
TRIM11-mediated ubiquitination of PAX6 promotes proteasomal degradation, regulating its turnover in retinal progenitors .
Product Specs
This PAX6 recombinant monoclonal antibody is produced using an in vitro expression system. The process involves cloning the PAX6 antibody DNA sequences from immunoreactive rabbits. The immunogen used is a synthesized peptide derived from the human PAX6 protein. The genes encoding the PAX6 antibodies are then inserted into plasmid vectors and these recombinant plasmid vectors are transfected into host cells to enable antibody expression. The PAX6 recombinant monoclonal antibody subsequently undergoes affinity-chromatography purification and is rigorously tested for functionality in ELISA, IHC, and FC applications. This ensures its reactivity with the human PAX6 protein.
PAX6 is a transcription factor that plays a critical role in the development of the eye, central nervous system, and other tissues. Its functions are essential for the formation and maintenance of various structures within the body. Dysregulation of PAX6 can lead to developmental disorders and vision-related conditions.
Target Background
- Our research has established that PAX6 influences cell cycle distribution and makes U251 cells more susceptible to oxidative stress. Notably, we discovered differences in the sensitivity to established chemotherapeutic drugs between PAX6 expressing (WT) cells and PAX6 knockout cells. PMID: 29716531
- A familial congenital cataract, coloboma, and nystagmus phenotype with variable expression caused by mutation in PAX6 in a South African family has been reported. PMID: 29930474
- PAX6 variants and deletions were identified in 94% of patients with aniridia from Australasia and Southeast Asia. PMID: 29618921
- High PAX6 expression is associated with retinoblastoma. PMID: 30015924
- Knock-in 2A-EGFP cassette into PAX6 exon of embryonic stem cells H1 with TALEN-based homology recombination could establish PAX6(WT/EGFP) H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. PMID: 29807013
- This study revealed an outstanding mutation pattern in non-coding PAX6 regions confirming that PAX6 remains the major gene for aniridia. PMID: 30291432
- This is the first report on the mutational spectrum of PAX6 in aniridia patients of Cypriot ancestry. PMID: 29901133
- Deletions of the 3' regulatory regions downstream of paired box protein (PAX6) abrogate its expression leading to aniridia due to PAX6 haploinsufficiency in a 44-year-old mother and her 13-year-old son. PMID: 29932076
- The results demonstrated that miR874 may serve tumor suppressive roles in pancreatic ductal adenocarcinoma (PDAC) by directly targeting PAX6. Therefore, miR874 may exhibit potential applications for treatment of patients with PDAC. PMID: 29845293
- PAX6 was upregulated in Colorectal cancer tissues and was negatively correlated with miR383 expression. PMID: 29512711
- PAX6 knockdown recapitulated effects similar to those observed following miR-655 overexpression regarding the proliferation, invasion and apoptosis of retinoblastoma (RB)cells. Rescue experiments demonstrated that restoration of PAX6 expression reversed the tumour-suppressing roles of miR-655 in RB cells PMID: 29436689
- Excessive PAX6 expression in insulin-challenged endometrial epithelial cells may contribute to the uncontrollable endometrial epithelial proliferation in polycystic ovarian syndrome PMID: 29112933
- Overexpression of CHD1L in embryonic cells upregulated the expression of ectoderm genes, especially PAX6 PMID: 28946814
- PAX6 mutations explained 96.7% of aniridia phenotypes in this study with only 3 of 91 probands lacking pathogenic variants in the gene. PMID: 28321846
- Mutation screening of PAX6 gene helped in identifying a novel heterozygous pathogenic variation g. 31801757dupG (c. 216-19dupG) that resulted in a frameshift mutation that extended into exon 7. PMID: 29380764
- It was suggested that miR-19, upregulated in osteosarcoma cells, negatively regulated the expression of Pax6, which can promote the malignant phenotypes of osteosarcoma cells via activation of the extracellular signal-regulated kinase signaling pathways PMID: 29345189
- Molecular genetic defects involving PAX6 were identified in 30 participants (91%), including 4 novel PAX6 mutations (Gly18Val; Ser65ProfsX14; Met337ArgfsX18; Ser321CysfsX34) and 4 novel chromosome 11p deletions inclusive of PAX6 or a known PAX6 regulatory region. PMID: 29217025
- Findings shed new light on the miR-223/PAX6 pathway in glioma and this pathway might modulate the sensitivity of glioma to TMZ via regulating PI3K/Akt signaling pathway. PMID: 28332226
- Our study identified two novel PAX6 variants in two families with aniridia and revealed the pathogenicity of the variants; this would expand the variant spectrum of PAX6 and help us better understand the molecular basis of aniridia, thus facilitating genetic counseling. PMID: 28157223
- Highly conserved Sox2/Pax6 bound site near the Sprouty2 locus was verified to promote cooperative dimerization designating Sprouty2 as a potential target reliant on Sox2/Pax6 cooperativity in several neural cell types. PMID: 29050852
- It is important to establish the molecular diagnosis early to avoid repeated and long-term screening for Wilms tumor. Our work further emphasizes that a wide range of ocular phenotypes are associated with loss of function PAX6 mutations. PMID: 26849621
- A novel mutation (c.1033-1_1033delinsCT) and a reported mutation (c.1183 + 1G > A) within PAX6 gene were identified in two families with aniridia. The PAX6 mutations caused aberrant patterns of RNA splicing. PMID: 28760551
- Reduced expression of PAX6 in metabolically stressed beta cells may contribute to beta cell failure and alpha cell dysfunction in diabetes. PMID: 27941241
- Variant aniridia, congenital iris ectropion associated with foveal hypoplasia in both a woman and her son with a mutation of PAX6 gene. To our knowledge, deletion c. 936delC in exon 8 of PAX6 gene has not been reported until now. PMID: 28300742
- Epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation PMID: 27583466
- PAX6-depleted CECs maintained the epithelial morphology, but became larger. Global analyses using microarray revealed that down-regulated genes were primarily CEC-specific PMID: 27818314
- Photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. PMID: 27229137
- Data indicate paired box gene 6 (aniridia, keratitis) protein (PAX6) haploinsufficiency as causal for aniridia. PMID: 26661695
- Downregulation of PAX6 is associated with glioma progression. PMID: 28184912
- Overexpression of paired box 6 increased the inhibitory effect of temozolomide on GBM cell proliferation; on the other hand, knockdown of paired box 6 abolished the inhibitory effect of temozolomide on GBM cell proliferation. PMID: 28035389
- The present study identified a heterozygous deletion and a run-on mutation in PAX6 in two families with autosomal dominant aniridia. PMID: 27431685
- Fourteen of these mutations presented in the known aniridia genes; PAX6, FOXC1 and PITX2. PMID: 27124303
- We show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression PMID: 26899008
- We report the first association study of the transcription factor PAX6 with HSCR and that its low expression levels may result in an aberrant neurogenesis, which is directly related with manifestation of HSCR phenotype. PMID: 26879676
- Data suggest that promoter hypermethylation of PAX6 is a common event in hepatocellular carcinoma and the association of PAX6 methylation in clinicopathological features is divergent with different viral status. PMID: 27110298
- Congenital primary aphakia is associated with PAX6 mutation. PMID: 26130484
- We found that PAX6 gene was specifically methylated in non small cell lung cancer PMID: 26617874
- Findings suggest that PAX6+/- is associated with smaller pineal size, lower melatonin secretion and greater parental report of sleep disturbances in children; further studies are needed to explore the potential use of melatonin replacement for improving sleep quality in patients with PAX6+/- PMID: 26439359
- In post-mortem substantia nigra from Parkinson's disease patients, a reduced number of PAX6 expressing cells were found. PMID: 26295830
- We analyzed the PAX6 gene in a Bosma arrhinia microphthalmia syndrome patient but found no variation or mutation that could constitute or establish a causal association in our patient PMID: 26440771
- Variants in TRIM44 Cause Aniridia by Impairing PAX6 Expression PMID: 26394807
- MicroRNA-7 inhibits the malignant phenotypes of nonsmall cell lung cancer in vitro by targeting Pax6 PMID: 26135959
- ChIP experiments confirmed that Pdx1 activates the expression of the downstream transcription factors, Ngn3 and Pax6, by combined with the promoter regions of insulin (Insulin-P), Ngn3 (Ngn3-P), and Pax6 (Pax6-P). PMID: 26345820
- We reported the results of the clinical and molecular evaluation of a three-generation Chinese family with aniridia and identified a rare heterozygous M1K mutation in PAX6. PMID: 26535646
- Trans-placental BPA exposure down-regulated gene expression of Sox2 and Pax6 potentially underlying the adverse effect on childhood neuronal development. [Meta-analysis] PMID: 25051057
- Molecular analysis identified two compound heterozygous TYR mutations known to cause OCAIA and cosegregate with oculocutaneous albinism. In addition, we identified a novel heterozygous PAX6 mutation confirming the atypical aniridia phenotype. PMID: 25687215
- SOX4, SOX11, and PAX6 were significant for tumor type. PMID: 25804118
- The case of a Korean family with a novel splice site mutation in the PAX6 gene in isolated aniridia inherited in an autosomal dominant manner, is reported. PMID: 25696017
- Loss of PAX6 converts LSCs to epidermal stem cells, as demonstrated by a switch in the keratin gene expression profile and by the appearance of congenital dermoid tissue PMID: 26045558
- Dkk3/REIC3 expression is regulated by PAX6 in several human cell lines. PMID: 25029272
Q&A
What is PAX6 and why is it significant in research?
PAX6 is a paired domain and homeodomain-containing transcription factor that plays crucial roles in development. It is one of the earliest genes expressed in the eye field and is considered a master control gene for retinal and eye development . Additionally, PAX6 is important in neurodevelopment and the differentiation of islet cells in the pancreas . Its significance stems from its conservation across species and its involvement in multiple developmental processes. Research into PAX6 contributes to our understanding of ocular disorders, neurological development, and various developmental pathways.
What distinguishes recombinant monoclonal antibodies from traditional monoclonal antibodies for PAX6 detection?
Recombinant monoclonal antibodies for PAX6 are generated through in vitro expression systems involving the cloning of PAX6 antibody DNA sequences from immunoreactive animals (typically rabbits) . The genes encoding these antibodies are inserted into plasmid vectors and transfected into host cells for expression. Unlike traditional hybridoma-derived antibodies, recombinant antibodies offer greater batch-to-batch consistency, specificity, and can be engineered for particular applications. For PAX6 research, this means more reliable detection across various experimental conditions and potentially higher specificity for particular PAX6 isoforms.
What are the optimal applications for PAX6 recombinant monoclonal antibodies?
PAX6 recombinant monoclonal antibodies are versatile and can be used across multiple experimental approaches. The most commonly validated applications include:
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Western Blot (WB): For quantitative analysis of PAX6 expression levels
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Immunohistochemistry (IHC): For localization studies in tissue sections
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Immunofluorescence (IF): For cellular localization and co-expression studies
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Flow Cytometry (FC): For quantitative analysis in cell populations
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ELISA: For quantitative detection in solution
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Chromatin Immunoprecipitation (ChIP): For studying PAX6 binding to DNA targets
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Immunoprecipitation (IP): For protein-protein interaction studies
More specialized applications include function blocking experiments and gel supershift assays for studying DNA-protein interactions .
What dilution protocols are recommended for different experimental applications?
Each antibody should be titrated in your specific experimental system to obtain optimal results .
How should researchers approach antigen retrieval for IHC with PAX6 antibodies?
For optimal IHC results with PAX6 antibodies, proper antigen retrieval is critical. The recommended protocol involves:
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Primary approach: Use TE buffer at pH 9.0 for heat-mediated antigen retrieval
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Alternative approach: Use citrate buffer at pH 6.0 if the primary method yields suboptimal results
The appropriate antigen retrieval method may vary based on tissue fixation conditions, tissue type, and the specific antibody used. Validation experiments comparing different antigen retrieval methods on your specific samples are recommended to determine optimal conditions.
What positive controls are recommended for validating PAX6 antibody performance?
When establishing a new experimental system, researchers should include at least one validated positive control sample alongside their experimental samples.
How should I design controls for experiments with PAX6 antibodies?
A robust experimental design for PAX6 antibody studies should include:
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Positive controls: Include tissues or cell lines with known PAX6 expression (see table above)
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Negative controls:
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Knockdown/knockout control: Where possible, include PAX6 knockdown or knockout samples
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Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm specificity
For developmental studies, include appropriate temporal controls, as PAX6 expression varies during development.
What species reactivity has been confirmed for PAX6 antibodies?
The DSHB PAX6 antibody shows exceptional cross-species reactivity, making it particularly valuable for evolutionary and comparative studies .
What considerations are important when using PAX6 antibodies across different species?
When using PAX6 antibodies across different species, researchers should consider:
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Epitope conservation: The DSHB PAX6 antibody targets the N-terminal region (aa 1-223), which is highly conserved across species
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Validation requirements: Even with predicted cross-reactivity, empirical validation is necessary
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Isoform differences: Different species may express different PAX6 isoforms, resulting in varied banding patterns
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Background concerns: Optimization of blocking and wash conditions may be needed for non-validated species
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Positive controls: Include samples from validated species alongside experimental samples from non-validated species
For evolutionary studies, the exceptional cross-species reactivity of the DSHB PAX6 antibody (which works in organisms from planaria to humans) makes it particularly valuable .
How can PAX6 antibodies be used to study developmental processes?
PAX6 antibodies are powerful tools for developmental biology research due to PAX6's role as a master regulator gene. Advanced applications include:
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Temporal expression mapping: Track PAX6 expression throughout developmental stages using IF or IHC
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Lineage tracing: Combine PAX6 immunostaining with other lineage markers to track cell fate
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Chromatin immunoprecipitation (ChIP): Identify PAX6 binding sites in the genome during development
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Function blocking: Use PAX6 antibodies with function-blocking capability to study developmental consequences of PAX6 inhibition
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Co-immunoprecipitation: Identify PAX6 interaction partners during different developmental stages
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Single-cell analysis: Combine with single-cell techniques to understand heterogeneity in PAX6 expression
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Organoid research: Study PAX6 in 3D organoid models of eye and brain development
Careful consideration of fixation methods is crucial for developmental tissues, as they can affect epitope accessibility.
What approaches are recommended for studying PAX6 isoforms?
PAX6 exists in multiple isoforms that may have distinct functions. Researchers studying PAX6 isoforms should consider:
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Antibody selection: Choose antibodies with epitopes that can distinguish between isoforms or that detect all relevant isoforms
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Western blot analysis: The DSHB PAX6 antibody detects multiple apparent molecular weights (38, 40, 46, 48 kDa), potentially corresponding to different isoforms
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RT-PCR: Complement antibody-based detection with isoform-specific primers
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Isoform-specific knockdown: Use siRNA targeting specific isoforms alongside antibody detection
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Recombinant expression: Express individual isoforms as positive controls
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Mass spectrometry: Use immunoprecipitation followed by mass spectrometry to identify specific isoforms present in samples
For comprehensive isoform analysis, researchers should combine antibody-based approaches with molecular techniques.
How should I troubleshoot weak or non-specific signals with PAX6 antibodies?
For Western Blot:
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Loading amount: Increase protein loading (PAX6 may be low abundance in some samples)
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Antibody concentration: Try higher primary antibody concentrations within the recommended range
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Exposure time: Increase exposure time for detection
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Blocking optimization: Test different blocking agents (BSA vs. milk)
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Membrane type: PVDF may provide better results than nitrocellulose for some antibodies
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Sample preparation: Ensure complete denaturation and reduction of samples
For IHC/IF:
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Antigen retrieval: Test both recommended methods (TE buffer pH 9.0 and citrate buffer pH 6.0)
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Antibody concentration: Use higher concentrations within the recommended range
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Incubation time: Extend primary antibody incubation (overnight at 4°C)
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Detection system: Use amplification systems (e.g., tyramide signal amplification)
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Autofluorescence: Use appropriate quenching methods if autofluorescence is an issue
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Fixation: Optimize fixation protocols, as overfixation can mask epitopes
What are the optimal storage and handling conditions for PAX6 antibodies?
For optimal antibody performance, aliquot upon first thaw to minimize freeze-thaw cycles, and store according to manufacturer recommendations. While many antibodies remain active at 4°C for extended periods, activity can vary significantly, making proper aliquoting and freezing the safest approach for valuable antibodies .
