DGKE Antibody

What is DGKE and why is it significant in biomedical research?

DGKE (Diacylglycerol kinase epsilon) is a 64 kDa protein involved in lipid metabolism that has gained significant attention due to its association with atypical hemolytic-uremic syndrome (aHUS), a rare but serious disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney failure . The protein belongs to the diacylglycerol kinase family and plays a critical role in cell signaling pathways by regulating the levels of diacylglycerol. DGKE is particularly important in research focused on thrombotic microangiopathies and kidney diseases, as recessive mutations in this gene have been identified as causative for some forms of aHUS that present in infancy . The study of DGKE through antibody-based techniques has become instrumental in understanding its expression patterns and functional implications in pathological states.

What types of DGKE antibodies are available for research applications?

Several types of DGKE antibodies are available for research purposes, primarily differentiated by their clonality and host species:

• Polyclonal antibodies: These are the most common type of DGKE antibodies, derived from rabbit hosts (e.g., Prestige Antibodies HPA017167, Novus Biologicals NBP1-59067) . Polyclonal antibodies recognize multiple epitopes on the DGKE protein, potentially providing stronger signals but with increased risk of non-specific binding.

• Monoclonal antibodies: Less common but available from providers such as Novus Biologicals (H00008526-M03) . These antibodies recognize a single epitope, offering higher specificity but potentially lower sensitivity compared to polyclonals.

The antibodies also differ in their target regions, with some targeting the N-terminal region of DGKE (e.g., NBP1-59067) and others targeting different parts of the protein. The selection of a particular antibody depends on the experimental application and specific research questions.

How do I select the appropriate DGKE antibody for my specific research application?

Selecting the appropriate DGKE antibody requires consideration of several experimental factors:

• Application compatibility: Review validated applications for each antibody. For instance, HPA017167 is validated for immunohistochemistry (1:50-1:200 dilution) and immunofluorescence (0.25-2 μg/mL) , while NBP1-59067 is primarily validated for Western blot applications .

• Species reactivity: Confirm that the antibody recognizes DGKE in your species of interest. The NBP1-59067 antibody, for example, reacts with both human and mouse DGKE , making it suitable for comparative studies across these species.

• Epitope specificity: Consider whether you need to target a specific region of DGKE. Some antibodies target the N-terminal region (e.g., NBP1-59067) , while others may recognize different domains.

• Validation status: Prioritize antibodies with published validation data. The top validated antibodies according to Antibodypedia include Proteintech Group 11900-1-AP (3 references), LSBio LS-C409303, and Novus Biologicals H00008526-M03 (1 reference) .

• Format requirements: Consider whether you need unconjugated antibodies or specific formats (e.g., BSA-free preparations for certain applications) .

For optimal selection, review the manufacturer's validation data, published literature using the antibody, and consider performing pilot experiments comparing multiple antibodies for your specific application.

What are the recommended protocols for Western blot analysis using DGKE antibodies?

For optimal Western blot results with DGKE antibodies, follow these methodological guidelines:

• Sample preparation:

  • Process fresh tissue or cell lysates in RIPA buffer with protease inhibitors

  • For DGKE detection, brain or lymph tissue samples can serve as positive controls

• Protein loading and separation:

  • Load 20-50 μg protein per lane

  • Use 8-10% SDS-PAGE gels for optimal separation of the 64 kDa DGKE protein

• Transfer and blocking:

  • Transfer to PVDF membrane (nitrocellulose is an acceptable alternative)

  • Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • For NBP1-59067: Use at 0.2-1 μg/ml concentration (approximately 1:500-1:2500 dilution)

  • For typical polyclonal DGKE antibodies: Start with 1:1000 dilution

  • Incubate overnight at 4°C with gentle rocking

• Detection and visualization:

  • Use appropriate HRP-conjugated secondary antibody (typically anti-rabbit IgG)

  • Develop using ECL substrate

  • Expected band size: approximately 64 kDa

• Troubleshooting:

  • If detecting multiple bands, consider longer blocking times and more stringent washing

  • For weak signals, extend primary antibody incubation time or increase concentration

This protocol has been optimized based on experimentally validated conditions for DGKE detection in human and mouse samples .

How should I optimize immunohistochemistry protocols for DGKE detection in tissue samples?

Optimizing immunohistochemistry (IHC) for DGKE requires attention to several methodological details:

• Tissue preparation and antigen retrieval:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Embed in paraffin and section at 4-5 μm thickness

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

• Blocking and antibody dilutions:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Apply protein block with 5% normal goat serum for 30 minutes

  • For HPA017167: Use at 1:50-1:200 dilution as recommended

  • Incubate primary antibody overnight at 4°C or 1 hour at room temperature

• Detection system:

  • Use polymer-based detection systems for enhanced sensitivity

  • Apply DAB chromogen and counterstain with hematoxylin

  • Mount with permanent mounting medium

• Controls and validation:

  • Include positive tissue controls (brain tissue is recommended)

  • Include a no-primary antibody negative control

  • Consider using tissues from DGKE knockout models as specificity controls if available

• Expected results:

  • DGKE typically shows cytoplasmic staining pattern

  • Expression varies across tissue types, with The Human Protein Atlas providing reference data for normal tissue distribution

This protocol is based on the manufacturer's recommendations for the Prestige Antibodies product line, which includes validated DGKE antibodies for immunohistochemistry applications .

What are the recommended methods for analyzing DGKE expression in patient samples with suspected genetic variants?

When analyzing DGKE expression in patient samples with suspected genetic variants, a multi-faceted approach is recommended:

• RNA analysis workflow:

  • Extract total RNA from peripheral blood leukocytes (known to express DGKE)

  • Perform RT-PCR using primers spanning critical exons (e.g., exons 4-6 for analyzing intronic mutations near exon 5)

  • Run electrophoresis to detect potential aberrant splice variants

  • Expected outcomes: Wild-type amplicon of 273 bp in normal samples; potential additional bands in samples with splicing mutations (e.g., ~300 bp, ~400 bp, and ~650 bp bands reported in cases with the c.888+40A>G mutation)

• Protein expression analysis:

  • Perform Western blot analysis using validated DGKE antibodies

  • Compare band patterns between patient and control samples

  • Look for truncated proteins or altered expression levels

• Immunohistochemistry in renal biopsies:

  • Use DGKE antibodies at recommended dilutions (1:50-1:200)

  • Compare staining patterns with control tissues

  • Document alterations in expression or localization

• Integrating genomic data:

  • Correlate expression findings with sequencing data

  • Pay special attention to intronic mutations that might affect splicing

  • Use in silico prediction tools like GenScan or Human Splicing Finder to predict effects of variants on splicing

This comprehensive approach has been successfully used to characterize novel DGKE mutations in patients with atypical hemolytic-uremic syndrome, including the identification of intronic mutations that lead to aberrant splicing .

What are the common challenges encountered when working with DGKE antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with DGKE antibodies:

• Non-specific binding and background issues:

  • Problem: High background or multiple non-specific bands in Western blots

  • Solution: Increase blocking time (2 hours at room temperature), use 5% BSA instead of milk for blocking, increase washing time and frequency (5×5 minutes with TBST), and optimize antibody concentration (start with higher dilutions of 1:1000-1:2000)

• Inconsistent or weak signal:

  • Problem: Low signal intensity despite confirmed DGKE expression

  • Solution: Increase protein load (50-75 μg), optimize antigen retrieval for IHC, extend primary antibody incubation time (overnight at 4°C), and use signal enhancement systems (amplification systems for IHC or more sensitive ECL substrates for WB)

• Batch-to-batch variability:

  • Problem: Different results with different antibody lots

  • Solution: Purchase larger quantities of a single lot for long-term studies, perform validation with each new lot, and include consistent positive controls

• Tissue-specific optimization challenges:

  • Problem: Protocols optimized for one tissue type fail in others

  • Solution: Adjust fixation times for different tissues, modify antigen retrieval conditions, and optimize antibody concentration for each tissue type

• Storage and stability issues:

  • Problem: Decreased antibody performance over time

  • Solution: Store at recommended temperature (-20°C), avoid repeated freeze-thaw cycles by preparing small aliquots, and add 50% glycerol for long-term storage (noting that glycerol may affect some applications)

Implementing these solutions has proven effective in optimizing DGKE antibody performance across various experimental contexts and tissue types.

How can I validate the specificity of DGKE antibodies for my experimental system?

Validating DGKE antibody specificity is crucial for experimental reliability. A comprehensive validation approach includes:

• Positive and negative control tissues:

  • Use known DGKE-expressing tissues (brain, lymph) as positive controls

  • Include tissues with minimal DGKE expression as negative controls

  • Compare staining patterns with published expression data

• Knockdown/knockout validation:

  • Perform siRNA knockdown of DGKE and confirm reduction in signal

  • Use CRISPR/Cas9-engineered DGKE knockout cells if available

  • Compare wild-type vs. modified samples in parallel experiments

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare results with and without peptide competition

  • Signal should be significantly reduced or eliminated with peptide competition

• Multiple antibody validation:

  • Use antibodies targeting different DGKE epitopes

  • Compare signal patterns across antibodies

  • Consistent patterns across different antibodies support specificity

• Molecular weight verification:

  • Confirm that the detected band matches the expected molecular weight (64 kDa for DGKE)

  • Analyze any additional bands for potential splice variants or post-translational modifications

• Mass spectrometry confirmation:

  • For definitive validation, immunoprecipitate with DGKE antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm DGKE presence and identify any co-precipitating proteins

This multi-faceted approach provides robust evidence for antibody specificity and ensures reliable experimental outcomes in DGKE research.

How do DGKE mutations contribute to the pathogenesis of atypical hemolytic-uremic syndrome (aHUS)?

DGKE mutations have emerged as significant genetic factors in atypical hemolytic-uremic syndrome (aHUS) pathogenesis through several molecular mechanisms:

• Disruption of endothelial thromboresistance:

  • DGKE normally phosphorylates diacylglycerol (DAG), converting it to phosphatidic acid

  • DGKE deficiency leads to DAG accumulation, enhancing protein kinase C activation

  • This results in increased endothelial activation and compromised thromboresistance

• Genetic inheritance pattern:

  • Unlike complement-mediated aHUS, DGKE-associated aHUS follows an autosomal recessive inheritance pattern

  • Both homozygous and compound heterozygous mutations can cause the disease

  • This explains familial clustering and early-onset presentation

• Clinical distinguishing features:

  • DGKE-aHUS typically presents in the first year of life

  • Patients experience multiple relapsing episodes

  • Proteinuria is a characteristic feature

  • These features are prototypical of DGKE-associated aHUS and differ from complement-mediated forms

• Molecular consequences of mutations:

  • Intronic mutations (e.g., c.888+40A>G) can create cryptic splice sites

  • This leads to aberrant mRNA transcripts with insertions or deletions

  • Resulting proteins may have altered catalytic domains or truncations

  • For example, the c.888+40A>G mutation produces three aberrant transcripts, including one that adds 13 amino acids to the protein (p.Lys296_Gly297ins13)

The understanding of DGKE-aHUS has advanced significantly through sequencing technologies and transcript analysis, revealing that even mutations outside traditional exonic regions can profoundly affect protein function and disease manifestation .

What are the latest findings on the role of DGKE in regulating thrombosis and inflammation?

Recent research has expanded our understanding of DGKE's role in thrombosis and inflammation regulation:

These findings highlight DGKE as a critical regulator at the intersection of thrombosis and inflammation, offering new therapeutic targets for DGKE-associated diseases beyond traditional complement-targeting approaches.

How can DGKE antibodies be utilized to study splicing defects and protein expression in patient samples?

DGKE antibodies provide powerful tools for investigating splicing defects and altered protein expression in patient samples:

• Combined transcript and protein analysis approach:

  • Start with RT-PCR analysis of DGKE transcripts to identify potential splicing abnormalities

  • Use DGKE antibodies in Western blot analysis to correlate transcript findings with protein expression

  • This integrated approach can reveal the consequences of genetic variants on both RNA processing and protein production

• Detection of abnormal protein products:

  • DGKE antibodies can detect aberrant protein products resulting from splicing defects

  • Western blot analysis can identify truncated proteins, extended proteins, or altered expression levels

  • For example, antibodies have been used to detect mutant DGKE proteins resulting from the c.888+40A>G mutation, which produces proteins with altered catalytic domains

• Tissue expression pattern analysis:

  • Immunohistochemistry with DGKE antibodies can map expression patterns in patient tissues

  • This can reveal altered subcellular localization or tissue-specific expression changes

  • For instance, kidney biopsies from aHUS patients can be examined for DGKE expression patterns

• Quantitative analysis techniques:

  • Use quantitative Western blot or ELISA with DGKE antibodies to measure expression levels

  • Compare expression between patient samples and controls

  • Correlate expression levels with disease severity and clinical manifestations

• Functional studies with immunoprecipitation:

  • DGKE antibodies can immunoprecipitate both wild-type and mutant proteins

  • The precipitated proteins can be used in kinase activity assays

  • This allows direct assessment of how splicing defects affect enzymatic function

This methodological framework has been successfully applied to characterize novel DGKE mutations, particularly those affecting splicing, and has contributed significantly to our understanding of genotype-phenotype correlations in DGKE-associated diseases .

What emerging applications of DGKE antibodies are being developed for diagnostic and research purposes?

Several innovative applications of DGKE antibodies are being developed that expand their utility in both diagnostics and research:

• Multiplexed tissue analysis:

  • Integration of DGKE antibodies into multiplexed immunofluorescence panels

  • Allows simultaneous detection of DGKE with complement proteins and endothelial markers

  • Provides spatial context for DGKE expression in relation to disease pathology

• Liquid biopsy applications:

  • Development of sensitive ELISA systems using DGKE antibodies

  • Potential for detecting DGKE protein fragments in plasma or urine

  • Could serve as non-invasive biomarkers for monitoring DGKE-associated diseases

• Single-cell analysis technologies:

  • Adaptation of DGKE antibodies for flow cytometry and mass cytometry (CyTOF)

  • Enables cell-specific analysis of DGKE expression in heterogeneous samples

  • Provides insights into which cell populations are most affected by DGKE mutations

• Therapeutic monitoring applications:

  • Using DGKE antibodies to monitor treatment responses

  • Assessment of DGKE expression or activity following experimental therapies

  • Potential development of companion diagnostics for future DGKE-targeted therapies

• High-throughput screening platforms:

  • Implementation of DGKE antibodies in protein array technologies

  • Facilitates screening of large patient cohorts for DGKE abnormalities

  • Enables population-level studies of DGKE in various diseases

These emerging applications hold promise for advancing both clinical diagnostics and basic research in DGKE-related pathologies, particularly in the context of rare diseases like atypical hemolytic-uremic syndrome.

How can researchers design experiments to investigate the relationship between DGKE and other signaling molecules in thrombotic microangiopathies?

Designing effective experiments to investigate DGKE's interactions with other signaling molecules in thrombotic microangiopathies requires sophisticated approaches:

• Co-immunoprecipitation studies:

  • Use DGKE antibodies to immunoprecipitate protein complexes

  • Analyze co-precipitated proteins by mass spectrometry

  • Confirm interactions with directed Western blots for suspected binding partners

  • This approach can identify novel interactions in the DGKE signaling network

• Proximity ligation assays:

  • Apply DGKE antibodies in combination with antibodies against potential interacting proteins

  • Visualize protein-protein interactions in situ with subcellular resolution

  • Quantify interaction frequencies under normal and disease conditions

• Phosphoproteomic analysis:

  • Compare phosphorylation profiles in normal vs. DGKE-deficient cells

  • Use phospho-specific antibodies to track activation of downstream pathways

  • Construct signaling network maps based on altered phosphorylation patterns

• CRISPR-based genetic screens:

  • Create DGKE knockout cell lines using CRISPR/Cas9

  • Perform genetic screens to identify synthetic lethal interactions

  • Validate findings with DGKE antibodies to confirm protein expression changes

• Ex vivo perfusion models:

  • Utilize patient-derived or genetically modified endothelial cells

  • Apply DGKE antibodies for immunofluorescence analysis after perfusion

  • Assess how DGKE deficiency affects thrombosis formation under flow conditions

• Experimental design table for DGKE signaling studies:

These methodological approaches provide complementary insights into DGKE's role in signaling networks relevant to thrombotic microangiopathies and can help identify novel therapeutic targets.

What are the most significant unanswered questions in DGKE research that antibody-based techniques could help address?

Several critical knowledge gaps in DGKE research could be addressed through advanced antibody-based techniques:

• Tissue-specific expression patterns and regulation:

  • Comprehensive mapping of DGKE expression across human tissues using validated antibodies

  • Investigation of how expression patterns change during development and aging

  • Identification of tissue-specific regulatory mechanisms controlling DGKE expression

• Post-translational modifications and their functional significance:

  • Development of modification-specific DGKE antibodies (phospho-, acetyl-, ubiquitin-specific)

  • Characterization of how post-translational modifications affect DGKE activity

  • Mapping of modification sites and their changes in disease states

• Subcellular localization dynamics:

  • Super-resolution microscopy with DGKE antibodies to track intracellular trafficking

  • Investigation of how mutations affect localization and membrane association

  • Identification of localization-dependent interaction partners

• Structural consequences of disease-associated mutations:

  • Epitope-specific antibodies to probe conformational changes caused by mutations

  • Analysis of how mutations affect protein stability and degradation rates

  • Investigation of potential dominant-negative effects of mutant proteins

• Biomarker potential in thrombotic microangiopathies:

  • Development of sensitive detection methods for circulating DGKE or its fragments

  • Correlation of DGKE levels or variants with disease progression and treatment response

  • Establishment of DGKE-based diagnostic criteria for different subtypes of thrombotic microangiopathies

Addressing these questions through antibody-based approaches would significantly advance our understanding of DGKE biology and its role in disease pathogenesis, potentially leading to new diagnostic and therapeutic strategies for DGKE-associated disorders.

How might current limitations in DGKE antibody technology be overcome in the future?

Current limitations in DGKE antibody technology could be addressed through several innovative approaches:

• Enhancing specificity and reducing cross-reactivity:

  • Development of monoclonal antibodies against underrepresented epitopes

  • Implementation of phage display technology to select high-specificity antibodies

  • Generation of recombinant antibody fragments with enhanced specificity

  • Using CRISPR knockout validation systems to ensure absolute specificity

• Improving detection of low-abundance DGKE variants:

  • Development of amplification systems for immunoassays

  • Implementation of single-molecule detection technologies

  • Creation of antibody-based proximity ligation assays for greater sensitivity

  • Integration with mass spectrometry for enhanced detection limits

• Addressing splice variant specificity:

  • Generation of isoform-specific antibodies targeting unique junction sequences

  • Development of antibody panels capable of distinguishing between normal and aberrant splice products

  • Implementation of RNA-protein co-detection methods

• Enhancing functional assessment capabilities:

  • Development of activity-state specific antibodies for DGKE

  • Creation of FRET-based antibody systems to detect DGKE conformational changes

  • Integration of antibodies with biosensors for real-time activity monitoring

• Expanding therapeutic applications:

  • Exploration of antibody-drug conjugates targeting cells with abnormal DGKE expression

  • Development of intrabodies capable of modulating DGKE function intracellularly

  • Creation of bispecific antibodies linking DGKE to degradation machinery