DHFS Antibody

What is DHPS/DHS and why is it important in research?

DHPS (Deoxyhypusine synthase) catalyzes the first step in the post-translational modification of eukaryotic initiation factor 5A (eIF-5A), specifically transferring the butylamine moiety of spermidine to the epsilon-amino group of a critical lysine residue in the eIF-5A precursor protein to form deoxyhypusine . This process is fundamental for the maturation of eIF-5A, which plays essential roles in cell proliferation, translation elongation, and mRNA decay. Studying DHPS is crucial for understanding protein synthesis regulation and may provide insights into cancer research, as eIF-5A is often dysregulated in malignancies. The DHPS antibody serves as an important tool for detecting and quantifying this enzyme in biological samples.

What are the typical applications for DHPS/DHS antibodies in research?

DHPS/DHS antibodies are primarily utilized in Western blotting (WB), immunohistochemistry on paraffin-embedded tissues (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF) . These applications enable researchers to detect and quantify DHPS protein expression in various human tissues and cell lines. The antibody allows for visualization of DHPS localization within cells and tissues, which is crucial for understanding its function in different cellular compartments. For example, DHPS antibodies have been validated for use in colon and prostate tissue samples , providing insights into the role of this enzyme in these specific tissues.

What is the molecular profile of human DHPS protein?

Human DHPS is typically observed at a molecular weight of approximately 39 kDa on Western blots, with a calculated molecular weight of 40,971 Da . The protein functions as a NAD-dependent enzyme and plays a critical role in the hypusination pathway. The commercially available antibodies are generally raised against the N-terminal portion (amino acids 1-150) of human DHPS . Understanding this molecular profile is essential for proper experimental design and interpretation of results when working with DHPS antibodies.

How do DHPS antibodies contribute to understanding post-translational modifications?

DHPS antibodies serve as crucial tools for elucidating the complex process of hypusination, a unique post-translational modification specific to eIF-5A. By employing these antibodies in combination with other molecular techniques, researchers can track the sequential enzymatic steps involving DHPS. This enzyme catalyzes the NAD-dependent oxidative cleavage of spermidine and transfers the butylamine moiety to the epsilon-amino group of a specific lysine residue in eIF-5A, forming the intermediate deoxyhypusine residue . Subsequent hydroxylation by deoxyhypusine hydroxylase (DOHH) completes the hypusination process. DHPS antibodies enable researchers to monitor this pathway's activity under various experimental conditions, providing insights into how alterations in hypusination affect cellular processes including protein synthesis, cell proliferation, and differentiation.

How can researchers optimize Western blotting protocols for DHPS detection?

For optimal Western blot results when detecting DHPS, researchers should consider several key parameters. Based on validation data, the recommended dilution range for DHPS antibodies in Western blotting applications is 1:500-1:2000 . K562 cells have been successfully used as positive controls in Western blot applications for DHPS antibody validation . The observed molecular weight of approximately 39 kDa should be used as a reference point for identifying the DHPS protein band. To enhance specificity and reduce background, researchers should optimize blocking conditions, antibody incubation times, and washing steps. Additionally, incorporating appropriate positive and negative controls is essential to ensure the validity of results and confirm antibody specificity.

What methodological approaches can be used to study DHPS interactions with eIF-5A?

To investigate the interactions between DHPS and eIF-5A, researchers can employ several sophisticated methodological approaches. Co-immunoprecipitation (Co-IP) using DHPS antibodies can capture protein complexes containing DHPS and its binding partners, including eIF-5A precursors. This can be followed by Western blotting to confirm the presence of specific interaction partners. For spatial resolution of these interactions, researchers can utilize immunofluorescence with dual labeling of DHPS and eIF-5A, examining their co-localization patterns in different cellular compartments or under various experimental conditions. Proximity ligation assays (PLA) offer another powerful approach for visualizing protein-protein interactions in situ with high sensitivity. Additionally, functional studies involving DHPS knockdown or inhibition can help elucidate the consequences of disrupted DHPS-eIF-5A interactions on hypusination and downstream cellular processes.

What are common challenges when using DHPS antibodies and how can they be addressed?

When working with DHPS antibodies, researchers frequently encounter several technical challenges. One common issue is non-specific binding, which can result in background signal and false positives. This can be mitigated by optimizing blocking conditions (using 3-5% BSA instead of milk for phosphorylated targets) and employing more stringent washing procedures. Another challenge is inconsistent detection across different cellular contexts, which may be addressed by adjusting antibody concentration based on expression levels in specific cell types. For tissues with high endogenous biotin, like kidney and liver, researchers should use avidin/biotin blocking kits to prevent non-specific binding when using biotin-based detection systems. Additionally, epitope masking in fixed tissues can occur; this may require testing different antigen retrieval methods (heat-induced versus enzymatic) to optimize signal detection. Storage conditions are also critical—DHPS antibodies should be stored at -20°C for long-term stability, while 4°C is suitable for short-term storage (up to one month) to avoid repeated freeze-thaw cycles that can degrade antibody quality .

How can researchers validate the specificity of DHPS antibodies in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable and reproducible results. For DHPS antibodies, researchers should implement a multi-faceted validation approach. First, positive control samples with known DHPS expression (such as K562 cells) should be used alongside experimental samples . Negative controls should include samples where DHPS is absent or knockdown/knockout models. Researchers can also perform peptide competition assays using the immunizing peptide (for example, a peptide derived from amino acids 51-100 of human DHPS) to confirm binding specificity . For advanced validation, siRNA or CRISPR-mediated knockdown/knockout of DHPS followed by antibody testing can provide compelling evidence of specificity. Different antibody lots should be compared to ensure consistent performance, and cross-reactivity with related proteins should be assessed, particularly in studies involving multiple species. Finally, complementary techniques (e.g., mass spectrometry) can be used to verify that the detected protein is indeed DHPS.

What are the optimal sample preparation methods for different DHPS antibody applications?

Optimal sample preparation varies considerably depending on the intended application:

For Western blotting: Cells or tissues should be lysed in a buffer containing protease inhibitors to prevent protein degradation. RIPA or NP-40 buffers are typically effective for DHPS extraction. Samples should be denatured at 95°C for 5 minutes in loading buffer containing SDS and a reducing agent. The recommended protein loading amount is 10-30 μg per lane.

For immunohistochemistry (IHC-P): Tissues should be fixed in 10% neutral-buffered formalin for 24-48 hours, followed by paraffin embedding and sectioning (4-6 μm thickness). Antigen retrieval is crucial—heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is often effective for DHPS detection. For DHPS antibodies that have been validated in human colon and prostate tissues, similar retrieval methods can be applied, with an optimal antibody dilution of 1:200 .

For immunocytochemistry/immunofluorescence (ICC/IF): Cells should be grown on coverslips, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.1-0.5% Triton X-100. Blocking with 1-5% BSA or normal serum is recommended before antibody incubation.

How should researchers interpret DHPS expression patterns across different tissues?

When analyzing DHPS expression patterns, researchers should consider tissue-specific variations that may reflect differing functional requirements for protein synthesis and cell proliferation. Based on immunohistochemical analyses, DHPS shows distinct expression patterns in human tissues, with notable presence in colon and prostate tissues . In rapidly dividing tissues, higher DHPS expression is typically observed, correlating with increased demand for eIF-5A hypusination to support protein synthesis. Researchers should compare their findings with established baseline expression data and consider both the intensity and subcellular localization of DHPS staining. Nuclear versus cytoplasmic localization may indicate different functional states or regulatory mechanisms. Additionally, changes in DHPS expression during development, disease progression, or in response to treatments should be interpreted in the context of the hypusination pathway's role in cell proliferation and protein synthesis. Quantitative analysis of staining intensity using digital image analysis can provide more objective assessments of expression levels across different samples.

What considerations are important when analyzing DHPS in relation to disease states?

When investigating DHPS in disease contexts, researchers must consider several critical factors. Altered DHPS expression or activity may reflect dysregulation of protein synthesis pathways, which is common in cancers and other proliferative disorders. Changes in DHPS levels should be correlated with patient clinical data, disease progression markers, and survival outcomes to establish clinical relevance. The relationship between DHPS and its substrate eIF-5A should be examined, as the ratio between these proteins may be more informative than absolute DHPS levels alone. Researchers should also consider post-translational modifications of DHPS itself, which might affect its activity independent of expression levels. Comparisons between diseased tissues and adjacent normal tissues from the same patient can control for individual variability. Finally, mechanistic studies using cell or animal models are essential to determine whether DHPS alterations are drivers or consequences of the disease process, which has implications for potential therapeutic targeting of this pathway.

How can researchers integrate DHPS antibody data with other molecular techniques for comprehensive pathway analysis?

For comprehensive analysis of the hypusination pathway, researchers should combine DHPS antibody-based detection with complementary molecular techniques. RNA-seq or qRT-PCR can be used to correlate DHPS protein levels with mRNA expression, revealing potential transcriptional or post-transcriptional regulatory mechanisms. Mass spectrometry-based approaches can identify post-translational modifications of DHPS or quantify hypusinated versus non-hypusinated eIF-5A. Metabolomic analysis of polyamine pathway components (putrescine, spermidine, spermine) can provide insights into substrate availability for DHPS activity. CRISPR-Cas9 gene editing or siRNA knockdown of DHPS followed by transcriptomic or proteomic profiling can reveal downstream effectors and cellular processes dependent on DHPS activity. Computational modeling integrating these multi-omics data can predict functional relationships and generate new hypotheses about DHPS regulation and function. This integrative approach enables researchers to place DHPS within its broader biological context, advancing understanding of how this enzyme contributes to normal physiology and disease states.

How might AI-driven approaches enhance DHPS antibody research?

Recent advancements in AI technology, such as the RFdiffusion model being developed for antibody design, offer promising approaches for DHPS research. AI models can be trained to design antibodies with enhanced specificity for different epitopes of DHPS or for detection of post-translationally modified variants of the protein . These computational methods could potentially overcome the challenges associated with traditional antibody development, which is often "challenging, slow, and expensive" . AI-driven antibody design could enable the creation of antibodies that specifically recognize the active versus inactive conformations of DHPS, or that can distinguish between different functional states related to NAD binding or substrate interaction. Additionally, computational models that integrate prediction of antibody-antigen binding with epitope immunogenicity could accelerate the development of both research tools and potential therapeutic antibodies targeting the DHPS pathway . Researchers should consider exploring these emerging technologies as they can significantly reduce the time and resources required for developing new, highly specific DHPS antibodies.

What are emerging applications of DHPS antibodies in therapeutic research?

As understanding of the hypusination pathway expands, DHPS antibodies are finding novel applications in therapeutic research. The essential role of DHPS in cell proliferation through the hypusination of eIF-5A makes it a potential target for anti-cancer therapies. Researchers are increasingly using DHPS antibodies to evaluate the efficacy of small molecule DHPS inhibitors, correlating inhibition of enzymatic activity with changes in protein expression and localization. These antibodies also enable the study of Fc-dependent mechanisms in therapeutic contexts, aligning with current research interests in antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) . Furthermore, DHPS antibodies are valuable tools for investigating the impact of hypusination inhibition on cancer cell survival, migration, and invasion. They can also help identify patient populations that might benefit from therapies targeting the hypusination pathway by enabling the analysis of DHPS expression patterns in tumor samples. This research direction could potentially lead to the development of companion diagnostics for hypusination-targeting therapeutics, representing an important bridge between basic research and clinical applications.

How can researchers address the technical limitations of current DHPS antibodies?

Current DHPS antibodies, while valuable, have several technical limitations that researchers are working to overcome. One significant challenge is the development of antibodies that can distinguish between the active (NAD-bound) and inactive conformations of DHPS. To address this, researchers are exploring the generation of conformation-specific antibodies using structural biology data to guide epitope selection. Another limitation is the restricted species reactivity of many DHPS antibodies (often human-specific) , which hinders comparative studies across model organisms. Developing cross-species reactive antibodies or species-specific panels would enable broader evolutionary and translational research. The sensitivity of detection in samples with low DHPS expression could be improved through signal amplification techniques such as tyramide signal amplification or polymer-based detection systems. Additionally, researchers are working on developing antibodies suitable for flow cytometry and ChIP applications, expanding the methodological toolkit for DHPS research. Collaborative efforts between academic labs and industry partners are essential for validating these new antibody tools across diverse experimental conditions and biological systems, ensuring their reliability for the research community.