HMGXB3 Antibody

What is HMGXB3 and what cellular functions does it regulate?

HMGXB3 belongs to the high-mobility group binding protein 3 family and is also known by synonyms HMGX3, KIAA0194, and SMF. This protein plays significant roles in cellular proliferation and migration pathways. Research has demonstrated that knockdown of HMGXB3 inhibits cell proliferation and reduces migration in gastric cancer cells and non-small cell lung cancer cells, suggesting its involvement in cancer progression mechanisms . At the molecular level, HMGXB3 forms a convergent gene pair with proto-oncogene c-fms, with only a 162 bp intergenic region between their 3′ ends. This arrangement facilitates the generation of mRNAs with extended 3′ ends that function as cis-antisense RNA against their partner mRNA, creating a natural regulatory system for controlling gene expression of both HMGXB3 and c-fms .

What are the optimal applications for HMGXB3 antibody in laboratory research?

HMGXB3 antibodies are primarily utilized in several molecular and cellular biology techniques, with the most validated applications being Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) . For Western Blotting applications, recommended dilution ratios typically range from 1:100-1:1000, providing sufficient sensitivity for detecting HMGXB3 protein in cellular lysates . ELISA applications generally require dilutions of 1:500-1:3000, depending on the specific assay format and detection system employed . Some HMGXB3 antibodies may also be applicable for Immunohistochemistry (IHC) and Immunofluorescence (IF) at dilutions of 1:100-1:500, though these applications should be validated independently for each specific antibody and experimental system . Researchers should note that HMGXB3 antibodies are furnished for laboratory research use only and should not be used in diagnostic, therapeutic, or other non-research applications .

What are the recommended storage and handling conditions for HMGXB3 antibody?

For optimal preservation of activity and specificity, HMGXB3 antibodies should be stored at -20°C in their supplied buffer, which typically consists of PBS with 0.1% Sodium Azide and 50% Glycerol at pH 7.3 . This formulation helps maintain antibody integrity during freeze-thaw cycles and provides stability during storage. To minimize potential degradation, it is advisable to aliquot the antibody upon first thawing to avoid repeated freeze-thaw cycles. When working with the antibody, it should be kept on ice and returned to -20°C storage promptly after use. The presence of sodium azide in the storage buffer serves as a preservative but should be noted as a potential inhibitor of horseradish peroxidase if used in enzyme-based detection systems. The 50% glycerol component acts as a cryoprotectant and helps prevent freeze damage to the antibody structure. For short-term usage (less than one week), storing at 4°C is acceptable, but prolonged storage at this temperature is not recommended as it may lead to reduced antibody performance over time.

What species reactivity does the HMGXB3 antibody demonstrate?

Most commercially available HMGXB3 antibodies are specifically designed to recognize human HMGXB3 antigens . These antibodies are typically developed using peptide antigens derived from human HMGXB3 sequences, making them highly specific for human samples in applications such as Western blotting and ELISA . When planning experiments involving multiple species, researchers should carefully verify the documented species reactivity of their chosen HMGXB3 antibody. While cross-reactivity with HMGXB3 from other species might occur due to sequence homology, this should be experimentally validated before use with non-human samples. Some specialized HMGXB3 antibodies may offer reactivity to other species, such as those derived from Borrelia burgdorferi , but these represent more specialized products for specific research applications. For comprehensive species reactivity information, researchers should consult the manufacturer's technical documentation and consider performing preliminary validation experiments when working with non-human samples.

What validation steps should be performed before using HMGXB3 antibody in critical experiments?

Before incorporating HMGXB3 antibody into critical experiments, a comprehensive validation protocol should be implemented to ensure specificity and optimal performance. Begin with Western blot analysis using positive control lysates from cell lines known to express HMGXB3 (based on literature or transcriptomic data) to confirm that the antibody detects a band of the expected molecular weight. Include negative controls such as lysates from HMGXB3 knockdown or knockout cells to demonstrate specificity. For more rigorous validation, conduct peptide competition assays where pre-incubation of the antibody with its immunizing peptide should abolish specific binding. For immunoassays such as ELISA, perform titration experiments with varying antibody concentrations to determine the optimal working range that provides the best signal-to-noise ratio. Cross-reactivity testing against related proteins, particularly other HMG box family members, should be performed to confirm specificity within this protein family. Additionally, verify lot-to-lot consistency when receiving new antibody batches by comparing performance against previous lots using standardized samples. Each validation step should be thoroughly documented according to good laboratory practice standards to support experimental reproducibility and publication requirements.

What dilution ratios are recommended for different applications of HMGXB3 antibody?

The optimal dilution ratios for HMGXB3 antibody vary by application technique and should be determined empirically for each experimental setup. Based on available technical data, the following recommendations can serve as starting points for optimization:

For each new lot of antibody or experimental system, it is advisable to perform a dilution series to identify the optimal concentration that provides the best signal-to-noise ratio . The antibody concentration of commercial HMGXB3 antibodies is typically around 200 μg/mL, which should be considered when calculating final working dilutions . Factors such as detection method sensitivity, sample preparation, and expression level of HMGXB3 in the experimental system will influence the optimal dilution, necessitating empirical determination for each specific application.

What sample preparation techniques optimize HMGXB3 antibody performance in different applications?

Optimal sample preparation for HMGXB3 antibody applications varies depending on the experimental technique and the cellular localization of the protein. For Western blotting, cells or tissues should be lysed in a buffer containing appropriate protease inhibitors to prevent HMGXB3 degradation. RIPA or NP-40 based buffers typically provide good results for extracting nuclear proteins like HMGXB3. When preparing cell lysates, it is advisable to include phosphatase inhibitors to preserve potential post-translational modifications that might affect antibody recognition. For protein extraction, sonication or mechanical disruption methods that efficiently disrupt nuclear membranes are recommended to ensure complete release of nuclear proteins.

For immunohistochemistry applications, formalin-fixed paraffin-embedded (FFPE) tissues typically require optimized antigen retrieval methods. Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine which provides optimal antigen accessibility while preserving tissue morphology. For immunofluorescence, paraformaldehyde fixation (4%) followed by permeabilization with 0.1-0.5% Triton X-100 is generally effective for nuclear proteins. In all immunological applications, thorough blocking with appropriate sera (5% BSA or 5-10% normal serum from the species of the secondary antibody) helps reduce background and improve signal specificity. Sample preparation should be consistent across experiments to ensure reproducibility, and appropriate positive and negative controls should be included to validate the preparation technique.

How can researchers troubleshoot non-specific binding when using HMGXB3 antibody?

Non-specific binding is a common challenge when working with polyclonal antibodies like HMGXB3. To address this issue, researchers should implement a systematic troubleshooting approach focusing on multiple aspects of the experimental protocol. First, optimize blocking conditions by testing different blocking agents (BSA, milk, normal sera) at various concentrations (3-10%) and increasing blocking time (1-2 hours at room temperature or overnight at 4°C). The choice of blocking agent can significantly impact background levels, with milk often providing better blocking for Western blots while BSA may be preferable for immunohistochemistry applications.

Increase the stringency of washing steps by using PBS with higher Tween-20 concentrations (0.1-0.5%) and extending wash durations. For Western blots specifically, consider adding 0.1-0.5% SDS to the antibody dilution buffer to enhance specificity. Reducing primary antibody concentration is often effective, as excessive antibody can lead to increased non-specific binding. Pre-absorbing the antibody with proteins from the species under investigation can help remove cross-reactive antibodies in the polyclonal mixture. Always include appropriate negative controls in each experiment, such as samples known not to express HMGXB3 or secondary antibody-only controls, to distinguish specific from non-specific signals. If problems persist despite these optimizations, consider testing alternative lots or suppliers of HMGXB3 antibody, as antibody quality can vary significantly between batches and manufacturers.

How can HMGXB3 antibody be used to investigate its role in cancer cell proliferation and migration?

HMGXB3 has emerged as a significant factor in cancer cell biology, with studies showing its involvement in proliferation and migration pathways. To investigate this role using HMGXB3 antibody, researchers can implement a multi-faceted experimental approach. First, establish baseline HMGXB3 expression across multiple cancer cell lines using Western blot with the antibody to identify suitable models with variable expression levels. Then, conduct siRNA or CRISPR-based knockdown/knockout of HMGXB3 followed by proliferation assays (MTT, BrdU incorporation) and migration assays (wound healing, transwell), using the antibody to confirm knockdown efficiency.

What experimental approaches can reveal the regulatory relationship between HMGXB3 and proto-oncogene c-fms?

The regulatory relationship between HMGXB3 and proto-oncogene c-fms represents a fascinating example of cis-antisense gene regulation. Research has revealed that these genes are positioned in a head-to-tail orientation with only a 162 bp intergenic region between their 3′ ends, facilitating the generation of mRNAs with extended 3′ ends that function as natural antisense transcripts . To investigate this relationship, researchers can employ several complementary approaches using HMGXB3 antibody as a key tool.

RNA stability assays following modulation of HMGXB3 expression are particularly informative. Experimental evidence shows that when HMGXB3 mRNA levels are reduced through miR-324-5p mimic treatment, c-fms mRNA stability dramatically increases, with its half-life extending from 8 hours to more than 24 hours . This suggests an inverse regulatory relationship that can be further explored through actinomycin D chase experiments following HMGXB3 knockdown or overexpression, with the antibody confirming protein-level changes.

RNA immunoprecipitation (RIP) using HMGXB3 antibody can identify whether HMGXB3 protein associates with c-fms mRNA or related regulatory complexes. Chromatin immunoprecipitation (ChIP) with HMGXB3 antibody might reveal whether HMGXB3 directly influences c-fms transcription through DNA binding. For visualizing the relationship, RNA FISH for both transcripts combined with immunofluorescence using HMGXB3 antibody can demonstrate spatial co-localization of these components within the cell. These approaches collectively provide a comprehensive toolkit for investigating this unique regulatory mechanism, with HMGXB3 antibody serving as an essential reagent for connecting RNA-level observations to protein function.

How does miR-324-5p regulation of HMGXB3 affect experimental approaches using the antibody?

miR-324-5p has been identified as a key post-transcriptional regulator of HMGXB3 expression through direct targeting of the 3′ UTR of HMGXB3 mRNA . This regulatory relationship has significant implications for experimental design when using HMGXB3 antibody. When implementing miR-324-5p manipulation as an experimental strategy to modulate HMGXB3 levels, researchers should consider several methodological approaches.

Treatment with miR-324-5p mimic dramatically reduces HMGXB3 mRNA stability, decreasing its half-life from 4 hours to less than 2 hours . This rapid degradation should be reflected at the protein level, which can be monitored by Western blotting with HMGXB3 antibody at various time points post-transfection. When designing such experiments, researchers should consider the protein's endogenous half-life to determine appropriate sampling times. The antibody can also be used to validate the efficacy of miR-324-5p inhibitors in rescuing HMGXB3 expression.

For mechanistic studies, luciferase reporter assays with wild-type and mutated HMGXB3 3′ UTR constructs can confirm direct miR-324-5p binding, while HMGXB3 antibody can be used in parallel Western blots to correlate reporter activity with endogenous protein levels. Interestingly, the same miR-324-5p mimic treatment that reduces HMGXB3 levels leads to increased stability of c-fms mRNA, the convergent gene partner of HMGXB3 . This inverse relationship can be investigated by dual immunoblotting for both proteins following miR-324-5p modulation, providing insight into their co-regulatory mechanism. When interpreting results from such experiments, researchers should be aware that changes in HMGXB3 expression induced by miR-324-5p may have secondary effects on other cellular pathways, requiring careful experimental design and appropriate controls.

What methods can be used to study HMGXB3 and c-fms sense-antisense RNA pairing mechanism?

The sense-antisense RNA pairing mechanism between HMGXB3 and c-fms represents a sophisticated example of natural antisense transcript (NAT) regulation. To study this complex mechanism, researchers can employ a combination of molecular and cellular techniques with HMGXB3 antibody serving as a critical reagent. RNA immunoprecipitation (RIP) using HMGXB3 antibody can pull down HMGXB3 protein complexes, which can then be analyzed for the presence of c-fms mRNA or regulatory RNAs involved in the sense-antisense pairing. This approach helps identify whether HMGXB3 protein directly interacts with the RNA regulatory complex.

For direct visualization of the sense-antisense RNA interaction, RNA fluorescence in situ hybridization (FISH) with differentially labeled probes for HMGXB3 and c-fms mRNAs can be combined with immunofluorescence using HMGXB3 antibody. This triple-labeling approach allows researchers to observe cellular co-localization of both transcripts and the HMGXB3 protein, providing spatial context for the regulatory mechanism. To investigate the functional consequences of this regulation, researchers can manipulate expression levels through siRNA knockdown of HMGXB3 followed by RNA stability assays for c-fms, using the antibody to confirm effective protein depletion .

Advanced techniques like Cross-Linking Immunoprecipitation (CLIP) combining UV cross-linking with immunoprecipitation using HMGXB3 antibody can provide single-nucleotide resolution of RNA-protein interactions. Structural studies through techniques like SHAPE (Selective 2′-hydroxyl acylation analyzed by primer extension) can elucidate the secondary structure of the sense-antisense RNA pairing region, complementing the protein-focused analyses enabled by the antibody. Together, these approaches provide a comprehensive toolkit for dissecting this unique regulatory mechanism at both the RNA and protein levels.

What are the differences between monoclonal and polyclonal HMGXB3 antibodies for research applications?

When selecting between monoclonal and polyclonal HMGXB3 antibodies, researchers should consider the distinct characteristics of each type and their experimental requirements. Currently available commercial HMGXB3 antibodies include polyclonal options derived from rabbit hosts . These polyclonal antibodies recognize multiple epitopes on the HMGXB3 protein, providing high sensitivity but potentially more background compared to monoclonal alternatives. Understanding the key differences between these antibody types is crucial for experimental design and result interpretation.

For applications requiring highest specificity and reproducibility (e.g., therapeutic development or standardized assays), monoclonal HMGXB3 antibodies would be preferred. For discovery research or applications where detecting low abundance targets is critical, polyclonal HMGXB3 antibodies may offer advantages due to their enhanced sensitivity. Validation for the specific intended application remains essential regardless of antibody type, with particular attention to controls that confirm specificity within the HMG protein family.

How do recombinant HMGXB3 proteins from different expression systems compare in research applications?

Recombinant HMGXB3 proteins can be produced in various expression systems, each conferring different characteristics that influence their utility in research applications. Based on available data, HMGXB3 proteins have been successfully expressed in mammalian systems such as HEK-293 cells and in yeast expression systems . The choice of expression system significantly impacts protein quality, post-translational modifications, yield, and suitability for downstream applications, which researchers should consider when selecting recombinant HMGXB3 proteins for their studies.

For applications requiring physiologically relevant HMGXB3 activity, such as protein-protein interaction studies or functional assays, mammalian-expressed HMGXB3 from HEK-293 cells is preferable despite higher costs . When using HMGXB3 as an immunogen or calibrator in ELISA assays, yeast-expressed protein may provide a cost-effective alternative with sufficient purity (>90%) . The expression system should be carefully matched to the research application, with consideration for required post-translational modifications, structural authenticity, and functional integrity. Researchers should also consider using the same recombinant protein source throughout a study to maintain consistency in experimental conditions and results.

What controls should be included when using HMGXB3 antibody in experimental studies?

Implementing robust controls is essential for generating reliable and reproducible results with HMGXB3 antibody. A comprehensive control strategy should address antibody specificity, experimental consistency, and biological relevance across different applications. The following controls should be considered mandatory for HMGXB3 antibody-based experiments:

• Positive Controls:

  • Lysates from cell lines with confirmed HMGXB3 expression

  • Recombinant HMGXB3 protein at known concentrations

  • Previously validated samples with established HMGXB3 expression patterns

• Negative Controls:

  • Samples from HMGXB3 knockout or knockdown systems

  • Cell lines with naturally low or undetectable HMGXB3 expression

  • Secondary antibody-only controls to assess non-specific binding

  • Isotype controls using matching IgG from the same species as the primary antibody (rabbit IgG for typical HMGXB3 antibodies)

• Specificity Controls:

  • Peptide competition assays using the immunizing peptide

  • Parallel testing with antibodies targeting different HMGXB3 epitopes

  • Cross-validation with alternative detection methods (e.g., mass spectrometry)

• Technical Controls:

  • Loading controls for Western blots (β-actin, GAPDH, or total protein stains)

  • Internal reference genes for qPCR validation of expression changes

  • Concentration gradients to demonstrate signal specificity and dynamic range

• Biological Controls:

  • Samples with experimentally modulated HMGXB3 expression

  • Related cell lines with different HMGXB3 expression profiles

  • Treatment conditions known to affect HMGXB3 expression (e.g., miR-324-5p modulation)

Thorough documentation of all controls and their results is essential for research transparency and reproducibility. This comprehensive control strategy helps distinguish genuine HMGXB3 signals from experimental artifacts and builds confidence in research findings, particularly when investigating novel functions or regulatory mechanisms of this relatively understudied protein.

How does HuR influence HMGXB3 expression through the c-fms regulatory pathway?

HuR (Human antigen R), an RNA binding protein that stabilizes target mRNAs, has been found to indirectly influence HMGXB3 expression through its direct interaction with c-fms mRNA . This regulatory relationship provides an interesting example of how RNA-binding proteins can exert broader influence beyond their direct targets through interconnected RNA networks. Understanding this mechanism is important for researchers studying HMGXB3 regulation and function.

The mechanism involves several distinct steps that have been experimentally verified. First, HuR binds directly to c-fms mRNA, as confirmed by immunoprecipitation assays showing a substantial 5.8-fold enrichment of c-fms mRNA in HuR immunoprecipitates compared to control IgG samples . In contrast, HMGXB3 mRNA shows no significant enrichment in HuR immunoprecipitates, indicating the absence of direct binding between HuR and HMGXB3 mRNA . The binding of HuR to c-fms mRNA stabilizes the transcript, increasing its half-life and steady-state expression levels within the cell.

Second, through the sense-antisense RNA pairing mechanism between c-fms and HMGXB3 mRNAs at their 3' ends, the increased stability and abundance of c-fms mRNA influences HMGXB3 mRNA levels . This influence operates through complementary base pairing at the 3' ends of the transcripts, where extended regions of the mRNAs can form cis-antisense interactions that affect stability and processing. The net effect is that HuR-mediated stabilization of c-fms mRNA indirectly impacts HMGXB3 expression, creating a regulatory cascade that extends HuR's influence to genes beyond its direct binding targets.

This regulatory pathway highlights the importance of considering indirect mechanisms when studying gene expression regulation. When investigating HMGXB3 expression patterns, researchers should account for factors affecting its sense-antisense partner c-fms, including RNA-binding proteins like HuR that may not directly interact with HMGXB3 mRNA but can significantly influence its expression through intermediate regulatory steps.