hoxa10b Antibody

What is HOXA10 and why is it significant in cancer research?

HOXA10 is a 410 amino acid sequence-specific transcription factor belonging to the Abd-B homeobox family, containing a homeobox DNA-binding domain. It functions primarily as part of a developmental regulatory system providing cells with specific positional identities on the anterior-posterior axis . HOXA10 has gained significant importance in cancer research due to its involvement in multiple malignancies. Studies have demonstrated that HOXA10 is highly expressed in several cancers and exhibits oncogenic activity . In gastric cancer, HOXA10 suppresses apoptosis and promotes proliferation, while facilitating cell proliferation and tumorigenesis through activation of the JAK1/STAT3 signaling pathway . Additionally, HOXA10 serves as a hub gene strongly associated with survival in pancreatic ductal adenocarcinoma (PDAC), functioning as a key regulator of immune suppression and stromal proliferation . Its involvement in multiple cellular processes, including proliferation, epithelial-mesenchymal transition (EMT), and chemotherapy resistance across various cancers, makes it a critical target for cancer research .

What is the difference between HOXA10 and HOXA10b antibodies?

HOXA10 refers to the human homeobox A10 protein, while HOXA10b typically designates a paralog found in certain model organisms such as zebrafish. The search results don't explicitly differentiate between HOXA10 and HOXA10b antibodies, but this distinction is important when selecting antibodies for cross-species studies . When choosing antibodies for research, scientists must carefully verify species reactivity, as antibodies raised against human HOXA10 may have varying degrees of cross-reactivity with HOXA10b from other species. Commercial antibodies like Proteintech's 26497-1-AP show confirmed reactivity with human samples and predicted reactivity with other mammals, but researchers working with zebrafish or other model organisms should perform validation studies to confirm cross-reactivity with HOXA10b in their specific model system .

What applications are HOXA10 antibodies typically used for in research?

HOXA10 antibodies are employed in a wide range of experimental applications for cancer and developmental biology research. According to available data, commercial HOXA10 antibodies have been validated for Western Blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), ELISA, and flow cytometry (FCM) applications . Western blotting is commonly used to detect HOXA10 protein expression levels, with the protein typically observed at 45-50 kDa despite its calculated molecular weight of 41 kDa . Immunohistochemistry applications include both paraffin-embedded (IHC-P) and frozen (IHC-F) tissue sections, allowing researchers to visualize HOXA10 expression patterns in tissues . Immunofluorescence techniques enable subcellular localization studies, particularly important since HOXA10 primarily localizes to the nucleus where it functions as a transcription factor . These varied applications make HOXA10 antibodies versatile tools for investigating this protein's role in various biological contexts.

What are the optimal conditions for using HOXA10 antibodies in Western blot experiments?

For optimal Western blot results with HOXA10 antibodies, researchers should carefully consider several methodological aspects. Based on commercial antibody specifications, a dilution range of 1:500-1:1000 is typically recommended for Western blot applications . Researchers should be aware that while the calculated molecular weight of HOXA10 is approximately 41 kDa, the observed molecular weight in Western blots typically appears between 45-50 kDa due to post-translational modifications . When designing experiments, positive controls such as HEK-293 or HeLa cell lysates are recommended as they have confirmed HOXA10 expression . For sample preparation, standard protocols including cell lysis with RIPA buffer supplemented with protease inhibitors are suitable. During optimization, researchers should test different blocking solutions (5% non-fat milk or BSA), incubation times, and washing stringency to minimize background. Additionally, validation of antibody specificity through knockout/knockdown experiments is strongly recommended, as published studies have used this approach to confirm specificity .

How should HOXA10 antibodies be validated for immunohistochemistry in cancer tissue samples?

Validating HOXA10 antibodies for immunohistochemistry (IHC) in cancer tissue samples requires a systematic approach to ensure specific and reproducible staining. First, researchers should conduct preliminary optimization experiments using both positive and negative control tissues. Based on published applications, antibodies like bs-2502R have been validated for both paraffin-embedded (IHC-P) and frozen (IHC-F) tissue sections . For antigen retrieval in formalin-fixed paraffin-embedded samples, citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine optimal conditions. Researchers should validate specificity through multiple methods: (1) comparison with mRNA expression data from the same tissue, (2) peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific staining, and (3) comparison of staining patterns between different HOXA10 antibodies targeting distinct epitopes . For gastric cancer tissues, where HOXA10 has been implicated in malignant phenotypes, validation can include correlation of staining intensity with clinical parameters such as tumor grade, stage, and patient survival, as HOXA10 expression correlates with poor prognosis in gastric cancer patients .

What controls are essential when studying HOXA10 in cancer cell lines?

When investigating HOXA10 in cancer cell lines, several essential controls must be incorporated to ensure experimental validity. First, researchers should include appropriate positive and negative control cell lines. For HOXA10 studies, HGC27 and AGS gastric cancer cell lines have been documented to express high levels of HOXA10, while GES-1 (normal human gastric mucosal epithelial cells) express lower levels, making these suitable comparative controls . Genetic manipulation controls are crucial: when performing knockdown experiments, multiple siRNAs targeting different regions of HOXA10 should be tested (e.g., si-HOXA10-AS#1, #2, and #3 as demonstrated in published work), and non-targeting siRNA controls (si-NC) must be included . For overexpression studies, empty vector controls are essential. Functional validation through rescue experiments provides powerful evidence of specificity – for example, demonstrating that phenotypes induced by HOXA10-AS can be reversed by HOXA10 knockdown . Additionally, researchers should verify HOXA10 expression changes at both mRNA (qRT-PCR) and protein (Western blot) levels following any experimental manipulation to ensure the observed phenotypes correlate with HOXA10 expression changes.

How can I differentiate between HOXA10 and HOXA10-AS when designing antibody-based experiments?

Differentiating between HOXA10 protein and HOXA10-AS (antisense long non-coding RNA) in antibody-based experiments requires careful experimental design due to their close genomic proximity and functional relationship. HOXA10-AS is transcribed antisense to HOXA10 and regulates HOXA10 expression, making their distinct detection critical for understanding their individual roles . For antibody-based experiments, it's important to recognize that antibodies will only detect the HOXA10 protein, not the HOXA10-AS lncRNA. To comprehensively study both molecules, researchers should employ a multi-technique approach: antibodies for detecting HOXA10 protein expression and localization, while RNA-based methods (qRT-PCR, RNA-FISH, or RNA-seq) should be used for HOXA10-AS detection . When interpreting results, researchers must consider their regulatory relationship—knockdown of HOXA10-AS has been shown to significantly reduce HOXA10 expression in gastric cancer cells . For mechanistic studies investigating how HOXA10-AS regulates HOXA10, researchers can design experiments where HOXA10-AS is knocked down or overexpressed, followed by assessment of HOXA10 protein levels using validated antibodies to establish the causative relationship observed in gastric cancer studies .

What strategies can be employed to study HOXA10 interactions with other proteins in cancer pathways?

Investigating HOXA10 protein interactions in cancer pathways requires sophisticated experimental approaches that extend beyond basic antibody applications. Co-immunoprecipitation (Co-IP) using HOXA10 antibodies represents a fundamental technique for identifying protein binding partners. For this application, antibodies must be carefully validated for immunoprecipitation efficiency, with protocols optimized for nuclear proteins since HOXA10 primarily localizes to the nucleus . Proximity ligation assays (PLA) offer an alternative in situ approach to visualize and quantify HOXA10 interactions with suspected partners like SIRT2, with which HOXA10 has been reported to interact . For a global interaction profiling approach, researchers can employ mass spectrometry following HOXA10 immunoprecipitation (IP-MS) to identify novel interaction partners. Chromatin immunoprecipitation (ChIP) using HOXA10 antibodies represents a critical technique for studying HOXA10's function as a transcription factor by identifying its genomic binding sites and target genes. Published studies in pancreatic cancer have identified HOXA10 as a master regulator controlling multiple genes, including BANF1, EIF4G1, MRPS10, PDIA4, and TYMS, making ChIP experiments valuable for confirming direct regulation . For functional validation of protein interactions, researchers should combine antibody-based detection methods with genetic manipulation approaches (CRISPR/Cas9 or siRNA) to demonstrate the biological significance of identified interactions in cancer-relevant phenotypes.

How can I accurately quantify HOXA10 expression in heterogeneous tumor samples?

Quantifying HOXA10 expression in heterogeneous tumor samples presents significant challenges that require specialized methodological approaches. Immunohistochemistry with HOXA10 antibodies offers spatial information about protein expression but requires standardized scoring systems. Researchers should employ digital pathology approaches with automated image analysis software to quantify staining intensity and percentage of positive cells across different regions of the tumor, minimizing subjective interpretation . For heterogeneous samples, multicolor immunofluorescence combining HOXA10 antibodies with markers for specific cell types (epithelial, stromal, immune) allows cell type-specific expression quantification. Laser capture microdissection followed by protein extraction and Western blot analysis provides another approach to analyze HOXA10 expression in specific tumor regions. Based on published PDAC studies, HOXA10 expression correlates with immunosuppression and tumor aggression, making it important to analyze HOXA10 expression in the context of tumor microenvironment by combining HOXA10 staining with immune cell markers . Validation of HOXA10 protein expression should be correlated with mRNA expression data, as demonstrated in studies where HOXA10 mRNA levels from TCGA databases were confirmed to correlate with protein expression in patient samples . For translational applications, researchers should consider developing standardized scoring systems for HOXA10 expression that can stratify patients into clinically relevant groups, as high HOXA10-AS expression has been associated with poor survival in gastric cancer patients .

Why might I observe discrepancies between HOXA10 mRNA and protein expression levels?

Discrepancies between HOXA10 mRNA and protein expression levels are frequently encountered in research and can result from multiple biological and technical factors. Post-transcriptional regulation represents a major biological factor, particularly relevant for HOXA10 as its expression can be regulated by HOXA10-AS, a long non-coding RNA that has been shown to promote HOXA10 expression in gastric cancer cells . This regulatory relationship may result in non-linear correlation between mRNA and protein levels. Post-translational modifications affecting protein stability may also contribute to discrepancies, as HOXA10 undergoes modifications that can influence its detection and half-life, evidenced by its observed molecular weight (45-50 kDa) being higher than its calculated weight (41 kDa) . Technical factors include antibody specificity issues, where antibodies may detect specific isoforms or modified versions of HOXA10, while mRNA detection methods capture all transcripts. Sampling differences between mRNA and protein analyses, particularly in heterogeneous tumors where different sections may be used for RNA extraction versus protein analysis, can also lead to apparent discrepancies. When encountering such discrepancies, researchers should verify results using multiple antibodies targeting different epitopes of HOXA10 and confirm protein expression in specific cell types using techniques like immunofluorescence combined with cell type-specific markers to understand the spatial context of expression patterns .

What are the common pitfalls when interpreting HOXA10 immunohistochemistry results in cancer tissues?

Interpreting HOXA10 immunohistochemistry results in cancer tissues presents several potential pitfalls that researchers must carefully navigate. Non-specific staining can result from improper antibody dilution or insufficient blocking, leading to false-positive interpretations. Researchers should validate staining patterns by comparing multiple HOXA10 antibodies and including appropriate negative controls such as isotype controls and tissues known not to express HOXA10 . Context-dependent expression patterns present another challenge, as HOXA10 expression varies across different cell types within the tumor microenvironment. Studies have shown that HOXA10 expression in PDAC is associated with immune cell infiltration patterns, specifically the proportion of T-cells and macrophages, highlighting the importance of analyzing HOXA10 expression in the context of the tumor microenvironment . Quantification inconsistencies arise from subjective scoring methods, particularly when comparing results across different studies or laboratories. Researchers should employ standardized scoring systems and, when possible, automated image analysis to minimize subjectivity . Clinical correlation pitfalls occur when attempting to associate HOXA10 expression with patient outcomes without considering other confounding factors. While high HOXA10-AS expression correlates with poor survival in gastric cancer patients, this relationship should be analyzed in multivariate models accounting for other prognostic factors like TNM stage and differentiation status, with which HOXA10-AS expression has been shown to correlate .

How can I reconcile contradictory findings about HOXA10 function across different cancer types?

Reconciling contradictory findings about HOXA10 function across different cancer types requires a systematic analytical approach considering multiple biological and experimental variables. Tissue-specific molecular contexts significantly influence HOXA10 function, as different tissues express distinct cofactors and downstream effectors that interact with HOXA10. In gastric cancer, HOXA10 promotes proliferation and suppresses apoptosis, while in pancreatic cancer, it is associated with immune suppression and enhanced tumorigenesis . Methodological differences across studies contribute to apparent contradictions; researchers should carefully compare experimental approaches, including cell lines used, knockdown efficiency, overexpression levels, and functional assays employed when comparing studies. The presence of different HOXA10 isoforms or post-translational modifications may result in distinct functional outcomes across cancer types. HOXA10's dual function as both a transcriptional activator and repressor depending on its binding partners may explain seemingly contradictory roles . When attempting to reconcile contradictory findings, researchers should characterize the specific HOXA10-dependent pathways active in their cancer model by performing comprehensive pathway analyses, similar to those conducted in PDAC studies that identified HOXA10 as a regulator of immune suppression . Integration of multi-omics data (transcriptomics, proteomics, epigenomics) can provide a more complete picture of HOXA10's context-dependent functions. Ultimately, validation experiments comparing HOXA10 function across multiple cancer types under identical experimental conditions may be necessary to directly address contradictory findings.

How might novel HOXA10 antibody technologies advance cancer biomarker development?

Emerging antibody technologies hold significant promise for advancing HOXA10-based cancer biomarker development. Single-domain antibodies (nanobodies) against HOXA10 could enable more sensitive detection in limited biopsy samples and potentially improve in vivo imaging of HOXA10-expressing tumors due to their small size and superior tissue penetration. Developing antibodies specifically recognizing post-translationally modified HOXA10 variants could reveal functionally distinct subpopulations associated with different cancer phenotypes, as HOXA10 undergoes modifications that may affect its oncogenic properties . Multiplexed antibody platforms combining HOXA10 detection with other cancer markers could create comprehensive diagnostic signatures. For instance, combining HOXA10 with the five-gene signature identified in pancreatic cancer (BANF1, EIF4G1, MRPS10, PDIA4, and TYMS) could improve prognostic accuracy for PDAC patients . For circulating tumor cell (CTC) detection, HOXA10 antibodies could be incorporated into microfluidic devices to identify CTCs with specific aggressive phenotypes, as HOXA10 is associated with malignant phenotypes in gastric cancer . HOXA10 antibodies conjugated to therapeutic agents represent another frontier, potentially enabling targeted delivery of cytotoxic drugs to HOXA10-overexpressing cancer cells. The development of antibodies capable of disrupting the regulatory relationship between HOXA10-AS and HOXA10 could offer novel therapeutic approaches for cancers where this interaction drives tumor progression .

How can HOXA10 antibody-based research contribute to precision medicine approaches for cancer treatment?

HOXA10 antibody-based research has significant potential to advance precision medicine for cancer treatment through several interconnected approaches. Development of companion diagnostic assays using standardized HOXA10 immunohistochemistry protocols could stratify patients for targeted therapies, particularly in gastric and pancreatic cancers where HOXA10 expression correlates with poor prognosis . These diagnostic applications would build on findings that high HOXA10-AS expression in gastric cancer patients correlates with poor survival and is associated with lymph node metastasis, TNM stage, and differentiation . Therapeutic resistance monitoring represents another application, as HOXA10 is involved in chemotherapy resistance mechanisms in various cancers . Periodic assessment of HOXA10 expression during treatment could identify emerging resistance, allowing timely therapeutic adjustments. For targeted therapy development, antibody-drug conjugates targeting HOXA10-expressing cells could deliver cytotoxic payloads specifically to cancer cells with high HOXA10 expression. Multi-marker prognostic panels incorporating HOXA10 with other molecular markers could enhance prediction accuracy for patient outcomes. The HOXA10-associated 5-gene signature (BANF1, EIF4G1, MRPS10, PDIA4, and TYMS) identified in PDAC represents a promising template for such approaches . Liquid biopsy applications could potentially detect HOXA10-expressing circulating tumor cells or extracellular vesicles containing HOXA10 protein, enabling non-invasive monitoring of disease progression. By integrating these antibody-based approaches with genomic and transcriptomic profiling, researchers can develop comprehensive molecular portraits of HOXA10-driven cancers, facilitating truly personalized therapeutic strategies targeting the specific molecular vulnerabilities of individual tumors.